Macrocyclic lactone compounds and methods for their use

ABSTRACT

The present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound of the formula: 
     
       
         
         
             
             
         
       
     
     wherein R 1 , R 2 , R 3 , R 5 , R 6 , R 8 , M 1 , M 2 , M 3 , M 4 , M 5 , M 6  and M 7  are each independently a member selected from the group consisting of H, C 1-6  alkyl, OH and C 1-6  hydroxyalkyl; R 4 , R 7  and R 9  are each independently selected from the group consisting of C 1-6  alkoxy and OH; R 10  is a member selected from the group consisting of H, —OH, —OP(O)Me 2 , 
     
       
         
         
             
             
         
       
     
     —O—(CH 2 ) n —OH and —O—(CH 2 ) m —O—(CH 2 ) o —CH 3 , wherein subscripts n and m are each independently from 2 to 8 and subscript o is from 1 to 6; each of L 1  and L 4  are independently selected from the group consisting of: 
     
       
         
         
             
             
         
       
     
     wherein each M 8  is independently a member selected from the group consisting of C 1-6  alkyl, OH and C 1-6  hydroxyalkyl; each of L 2  and L 3  are independently selected from the group consisting of: 
     
       
         
         
             
             
         
       
     
     and salts, hydrates, isomers, metabolites, N-oxides and prodrugs thereof.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/046,024, filed Mar. 11, 2008, which claims the benefit of U.S.application Ser. No. 11/854,312, filed Sep. 12, 2007, which claims thebenefit of U.S. Provisional Application No. 60/825,531, filed Sep. 13,2006, the full disclosures of which are incorporated by reference hereinin their entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

Not Applicable

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

Not Applicable

FIELD OF THE INVENTION

The present invention relates to structures of demethyl, hydroxyl,demethylhydroxyl, epoxide, N-oxide, opened hemiketal ring andseco-macrocyclic lactones, their synthesis, pharmaceutical compositionsand use for systemic and site specific therapeutic applications.

BACKGROUND

Rapamycin (Sirolimus) is a 31-member natural macrocyclic lactone[C51H79N1O13; MWt=914.2] produced by Streptomyces hygroscopicus andfound in the 1970s (U.S. Pat No. 3,929,992 ; 3,993,749). Rapamycin(structure shown below) was approved by the Food and Drug Administration(FDA) for the prophylaxis of renal transplant rejection in 1999.

Rapamycin resembles tacrolimus (binds to the same intracellular bindingprotein or immunophilin known as FKBP-12) but differs in its mechanismof action. Whereas tacrolimus and cyclosporine inhibit T-cell activationby blocking lymphokine (e.g., IL2) gene transcription, sirolimusinhibits T-cell activation and T lymphocyte proliferation by binding tomammalian target of rapamycin (mTOR). Rapamycin can act in synergy withcyclosporine or tacrolimus in suppressing the immune system.

Rapamycin is also useful in preventing or treating systemic lupuserythematosus [U.S. Pat. No. 5,078,999], pulmonary inflammation [U.S.Pat. No. 5,080,899], insulin dependent diabetes mellitus [U.S. Pat. No.5,321,009], skin disorders, such as psoriasis [U.S. Pat. No. 5,286,730],bowel disorders [U.S. Pat. No. 5,286,731], smooth muscle cellproliferation and intimal thickening following vascular injury [U.S.Pat. Nos. 5,288,711 and 5,516,781], adult T-cell leukemia/lymphoma[European Patent Application 525,960 A1], ocular inflammation [U.S. Pat.No. 5,387,589], malignant carcinomas [U.S. Pat. No. 5,206,018], cardiacinflammatory disease [U.S. Pat. No. 5,496,832], anemia [U.S. Pat. No.5,561,138] and increase neurite outgrowth [Parker, E. M. et al,Neuropharmacology 39, 1913-1919, 2000].

Although rapamycin can be used to treat various disease conditions, theutility of the compound as a pharmaceutical drug has been limited by itsvery low and variable bioavailability and its high immunosuppressivepotency and potential high toxicity. Also, rapamycin is only veryslightly soluble in water. To overcome these problems, prodrugs andanalogues of the compound have been synthesized. Water soluble prodrugsprepared by derivatizing rapamycin positions 31 and 42 (formerlypositions 28 and 40) of the rapamycin structure to form glycinate,propionate, and pyrrolidino butyrate prodrugs have been described (U.S.Pat. No. 4,650,803). Some of the analogues of rapamycin described in theart include monoacyl and diacyl analogues (U.S. Pat. No. 4,316,885),acetal analogues (U.S. Pat. No. 5,151,413), silyl ethers (U.S. Pat. No.5,120,842), hydroxyesters (U.S. Pat. No. 5,362,718), as well as alkyl,aryl, alkenyl, and alkynyl analogues (U.S. Pat. Nos. 5,665,772;5,258,389; 6,384,046; WO 97/35575).

Prodrugs and analogues of rapamycin are synthesized by chemicalsynthesis, where additional synthetic steps are required to protect anddeprotect certain positions. Analogues can also be synthesizedbiologically, where the Streptomyces strain is genetically modified toproduce these analogues of rapamycin. The analogues need to maintainnecessary positions for protein binding or other cellular interactionsand not generate steric hindrance in order to preserve its activity. Thesafety of these analogues requires extensively testing by series ofpreclinical and clinical experimentations.

The present invention comprises novel macrocyclic lactones and noveluses for macrocyclic lactones, where the compositions can be synthesizedchemically or biologically and which preserve at least someimmunosuppressive, anti-proliferative, anti-fungal and anti-tumorproperties for use in systemic and site specific applications.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising a pharmaceutically acceptable excipient and acompound of the formula:

wherein R¹, R², R³, R⁵, R⁶, R⁸, M¹, M², M³, M⁴, M⁵, M⁶ and M⁷ are eachindependently a member selected from the group consisting of H, C₁₋₆alkyl, OH and C₁₋₆ hydroxyalkyl; R⁴, R⁷ and R⁹ are each independentlyselected from the group consisting of C₁₋₆ alkoxy and OH;

R¹⁰ is a member selected from the group consisting of H, —OH, —OP(O)Me₂,

—O—(CH₂)_(n)—OH and —O—(CH₂)_(m)—O—(CH₂)_(o)—CH₃, wherein subscripts nand m are each independently from 2 to 8 and subscript o is from 1 to 6;each of L¹ and L⁴ are independently selected from the group consistingof:

wherein each M⁸ is independently a member selected from the groupconsisting of C₁₋₆ alkyl, OH and C₁₋₆ hydroxyalkyl; each of L² and L³are independently selected from the group consisting of:

and salts, hydrates, isomers, metabolites, N-oxides and prodrugsthereof.

In a second embodiment, the present invention provides a device forintracorporeal use, the device comprising an implant; and at least onesource of a compound of the present invention.

In a third embodiment, the present invention provides a method ofinhibiting cell proliferation by administering to a subject in needthereof, a therapeutically effective amount of a compound of the presentinvention.

In a fourth embodiment, the present invention provides macrocycliclactone compounds of the following formula:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, M¹, M², M³, M⁴, M⁵, M⁶,M⁷, L¹, L², L³ and L⁴ are as described above;

with the proviso that when R¹, R⁶, R⁸, M⁶ and M⁷ are Me, R³, R⁵, M¹, M²,M³, M⁴ and M⁵ are H, R⁴, R⁷ and R⁹ are OMe, R¹⁰ and M⁸ are OH, L² and L³are —CH═CH—, and L¹ and L⁴ are

R² is other than OH;

with the proviso that when R¹,R⁶, R⁸, M⁶ and M⁷ are Me, R², R³, R⁵, M¹,M², M³, M⁴ and M⁵ are H, R⁷ and R⁹ are OMe, R¹⁰ and M⁸ are OH, L² and L³are —CH═CH—, and L¹ and L⁴ are

R⁴ is other than OH;

with the proviso that when R¹, R⁶, R⁸, M⁶ and M⁷ are Me, R², R³, R⁵, M¹,M², M³, M⁴ and M⁵ are H, R⁴ and R⁷ are OMe, R¹⁰ and M⁸ are OH, L² and L³are —CH═CH—, and L¹ and L⁴ are

R⁹ is other than OH;

with the proviso that when R¹, R⁶, R⁸, M⁶ and M⁷ are Me, R², R³, R⁵, M¹,M², M³, M⁴ and M⁵ are H, R⁴, R⁷ and R⁹ are OMe, R¹⁰ and M⁸ is OH, L² andL³ are —CH═CH—, and L¹ and L⁴ are

R¹⁰ is other than OH, —OP(O)Me₂,

—O—(CH₂)_(n)—OH and —O—(CH₂)_(m)—O—(CH₂)_(o)—CH₃; and salts, hydrates,isomers, metabolites, N-oxides and prodrugs thereof.

In a fifth embodiment, the present invention provides a method of makinga compound of the present invention, the method comprising contacting amacrocyclic lactone with an acid to replace an alkoxy group with anucleophile, thereby making a compound of the present invention.

In a sixth embodiment, the present invention provides a method of makinga compound of the present invention, the method comprising contacting amacrocyclic lactone with an epoxidation agent to modify an alkene groupto an epoxide, thereby making a compound of the present invention.

In a seventh embodiment, the present invention provides a pharmaceuticalcomposition including a pharmaceutically acceptable excipient and acompound of the formula:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, M¹, M², M³, M⁴, M⁵, M⁶,M⁷, L¹, L², L³ and L⁴ are as described above; and salts, hydrates,isomers, metabolites, N-oxides and prodrugs thereof.

In an eighth embodiment, the present invention provides a pharmaceuticalcomposition including a pharmaceutically acceptable excipient and acompound of the formula:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, M¹, M², M³, M⁴, M⁵, M⁶,M⁷, L¹, L², L³ and L⁴ are as described above; and salts, hydrates,isomers, metabolites, N-oxides and prodrugs thereof.

In a ninth embodiment, the present invention provides a method oftreating an ophthalmic condition or disease by administering to asubject in need thereof, a therapeutically effective amount of acompound of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of macrocyclic lactones with some potentialsites for chemical modifications to provide compounds of the presentinvention. Areas marked with a square are sites for demethylation, andareas marked by A circle are sites for hydroxylation and C═C sites(C17═C18, C19═C20═, C21═C22, C29═C30) are sites for epoxidation.

FIG. 2A-2FV shows compounds of the present invention.

FIG. 3 shows an example of stent configuration having an expandablestructure.

FIG. 4 shows a preparative HPLC chromatogram of Compound AR.

FIG. 5 shows a Proton-NMR spectra of Compound AR.

FIG. 6 shows results of liquid chromatography and mass spectra ofCompound AR.

FIG. 7( a) shows an analytical HPLC chromatogram of Compound AR.

FIG. 7( b) shows an analytical HPLC chromatogram of Compound AR withisomers.

FIG. 8 shows percentage proliferation of human smooth muscle cells afterexposure to varying concentrations of rapamycin and Compound AR.

FIG. 9 shows quantitative coronary angiography (QCA) stenosis of theCompound AR eluting stent as compared to rapamycin eluting Cypher stentafter 28 day implantation in a porcine coronary artery model.

FIG. 10 shows tissue concentration of Compound AR at different timepoints in a porcine coronary artery model.

FIG. 11 shows percentage of Compound AR released from the stent in aporcine coronary artery model.

FIG. 12( a) shows inhibition of IL-6, MMP-9 and MCP-1 released byactivated macrophages by exposure to macrocyclic lactone Compound AR andSirolimus at 10 nM concentration.

FIG. 12( b) shows inhibition of IL-10 released by activated macrophagesby exposure to macrocyclic lactone Compound AR and Sirolimus at 10 nMconcentration

FIG. 13 shows percentage proliferation of human smooth muscle cellsafter exposure to varying concentrations of 17,18-29,30-bis-epoxidemacrocyclic lactone and Compound AR.

FIG. 14 shows the synthesis of 16-O-demethyl macrocyclic lactone of thepresent invention.

FIG. 15 shows the synthesis of 19, 20 bis-hydroxy macrocyclic lactone.

FIG. 16 shows the synthesis of 17,18-29,30-bis epoxide macrocycliclactone.

FIG. 17 shows the synthesis of 31-hydroxyl, 44-hydroxyl and 47-hydroxylmacrocyclic lactones.

FIG. 18 shows the synthesis of 43-hydroxyl and 47-hydroxyl macrocycliclactones.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

As used herein, the term “acid” refers to any chemical compound that,when dissolved in water, gives a solution with a pH less than 7.0. Acidsare generally described as a compound which donates a hydrogen ion (H+)(Bronsted-Lowry) or as an electron-pair acceptor (Lewis acid). Acidsuseful in the present invention include, but are not limited to, HCl,H₂SO₄, HNO₃ and acetic acid. One of skill in the art will appreciatethat other acids are useful in the present invention.

As used herein, “administering” refers to systemic and localadministration or a combination thereof such as oral administration,administration as a suppository, topical contact, parenteral,intravascular, intravenous, intraperitoneal, intramuscular,intralesional, intranasal, pulmonary, mucosal, transdermal, subcutaneousadministration, intrathecal, intraocular, intravitreal administration,delivery through a temporary device such as catheter, porous balloon,delivery through implant such as polymeric implant, osmotic pump,prosthesis such as drug eluting stents or others to the subject. One ofskill in the art will appreciate that other modes and methods ofadministering the compounds of the present invention are useful in thepresent invention.

As used herein, the term “alkoxy” refers to alkyl with the inclusion ofan oxygen atom, for example, methoxy, ethoxy, etc.“Halo-substituted-alkoxy” is as defined for alkoxy where some or all ofthe hydrogen atoms are substituted with halogen atoms. For example,halo-substituted-alkoxy includes trifluoromethoxy, etc. One of skill inthe art will appreciate that other alkoxy groups are useful in thepresent invention.

As used herein, the term “alkyl” refers to a straight or branched,saturated, aliphatic radical having the number of carbon atomsindicated. For example, C₁-C₆ alkyl includes, but is not limited to,methyl, ethyl, propyl, butyl, pentyl, hexyl, iso-propyl, iso-butyl,sec-butyl, tert-butyl, etc. One of skill in the art will appreciate thatother alkyl groups are useful in the present invention.

As used herein, the term “hydroxyalkyl” refers to alkyl as defined abovewhere at least one of the hydrogen atoms is substituted with a hydroxygroup. For example, hydroxyalkyl includes hydroxy-methyl, hydroxy-ethyl(1- or 2-), hydroxy-propyl (1-, 2- or 3-), hydroxy-butyl (1-, 2-, 3- or4-), hydroxy-pentyl (1-, 2-, 3-, 4- or 5-), hydroxy-hexyl (1-, 2-, 3-,4-, 5- or 6-), 1,2-dihydroxyethyl, and the like. One of skill in the artwill appreciate that other hydroxyalkyl groups are useful in the presentinvention.

As used herein, the term “body lumen” refers to the lining or cavity ofan artery, vein or an organ.

As used herein, the term “contacting” refers to the process of bringinginto contact at least two distinct species such that they can react. Itshould be appreciated, however, that the resulting reaction product canbe produced directly from a reaction between the added reagents or froman intermediate from one or more of the added reagents which can beproduced in the reaction mixture.

As used herein, the term “hydrate” refers to a compound that iscomplexed to at least one water molecule. The compounds of the presentinvention can be complexed with from 1 to 100 water molecules.

As used herein, the term “implant” refers to a medical device insertedinto a body in order to treat a condition. Implants include, but are notlimited to, drug-eluting devices.

As used herein, the terms “inhibition”, “inhibits” and “inhibitor” referto a compound that prohibits, reduces, diminishes or lessens, or to amethod of prohibiting, reducing, diminishing or lessening a specificaction or function.

As used herein, the term “intracorporeal” refers to an mammalian body.

As used herein, the term “isomer” refers to compounds of the presentinvention that possess asymmetric carbon atoms (optical centers) ordouble bonds, the racemates, diastereomers, enantiomers, geometricisomers, structural isomers and individual isomers are all intended tobe encompassed within the scope of the present invention.

As used herein, the term “organ” refers to any organ of a mammal, suchas, but not limited to, heart, lungs, brain, eye, stomach, spleen,bones, pancreas, kidneys, liver, intestines, uterus, colon, ovary,blood, skin, muscle, tissue, prostate, mammary and bladder. One of skillin the art will appreciate that other organs are useful in the presentinvention.

As used herein, the term “peracid” refers to an acid in which an acidic—OH group has been replaced by an —OOH group. Peracids can beperoxy-carboxylic acids of the formula R—C(O)—OOH, where the R group canbe groups such as H, alkyl, alkene or aryl. Peracids include, but arenot limited to, peroxy-acetic acid and meta-chloro-peroxybenzoic acid(MCPBA). One of skill in the art will appreciate that other peracids areuseful in the present invention.

As used herein, the term “peroxide” refers to a compound containing anoxygen-oxygen single bond. Examples of peroxides include, but are notlimited to, hydrogen peroxide. One of skill in the art will appreciatethat other peroxides are useful in the present invention.

As used herein, the term “pharmaceutically acceptable excipient” refersto a substance that aids the administration of an active agent to andabsorption by a subject. Pharmaceutical excipients useful in the presentinvention include, but are not limited to, polymers, solvents,antioxidants, binders, fillers, disintegrants, lubricants, coatings,sweeteners, flavors, stabilizers, colorants, metals, ceramics andsemi-metals. See below for additional discussion of pharmaceuticallyacceptable excipients. One of skill in the art will recognize that otherpharmaceutical excipients are useful in the present invention.

As used herein, the term “polymer” refers to a molecule composed ofrepeating structural units, or monomers, connected by covalent chemicalbonds. Polymers useful in the present invention are described below. Oneof skill in the art will appreciate that other polymers are useful inthe present invention.

As used herein, the term “prodrug” refers to compounds which are capableof releasing the active agent of the methods of the present invention,when the prodrug is administered to a mammalian subject. Release of theactive ingredient occurs in vivo. Prodrugs can be prepared by techniquesknown to one skilled in the art. These techniques generally modifyappropriate functional groups in a given compound. These modifiedfunctional groups however regenerate original functional groups byroutine manipulation or in vivo. Prodrugs of the active agents of thepresent invention include active agents wherein a hydroxy, amidino,guanidino, amino, carboxylic or a similar group is modified.

As used herein, the term “salt” refers to acid or base salts of thecompounds used in the methods of the present invention. Illustrativeexamples of pharmaceutically acceptable salts are mineral acid(hydrochloric acid, hydrobromic acid, phosphoric acid, and the like)salts, organic acid (acetic acid, propionic acid, glutamic acid, citricacid and the like) salts, quaternary ammonium (methyl iodide, ethyliodide, and the like) salts. Additional information on suitablepharmaceutically acceptable salts can be found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, which is incorporated herein by reference.

Pharmaceutically acceptable salts of the acidic compounds of the presentinvention are salts formed with bases, namely cationic salts such asalkali and alkaline earth metal salts, such as sodium, lithium,potassium, calcium, magnesium, as well as ammonium salts, such asammonium, trimethyl-ammonium, diethylammonium, andtris-(hydroxymethyl)-methyl-ammonium salts.

Similarly acid addition salts, such as of mineral acids, organiccarboxylic and organic sulfonic acids, e.g., hydrochloric acid,methanesulfonic acid, maleic acid, are also possible provided a basicgroup, such as pyridyl, constitutes part of the structure.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

As used herein, the term “source” refers to a location on the device ofthe present invention providing a supply of the compound of the presentinvention or a supply of a therapeutic agent. The device of the presentinvention can have more than one source, such as a first and second.Each source can have a different compound and composition and be used totreat a different indication.

As used herein, the term “subject” refers to animals such as mammals,including, but not limited to, primates (e.g., humans), cows, sheep,goats, horses, pigs, dogs, cats, rabbits, rats, mice and the like. Incertain embodiments, the subject is a human.

As used herein, the term “therapeutic agent” refers to any agent,compound or biological molecule that has a therapeutic effect on thepatient to whom the therapeutic agent is administered.

As used herein, the terms “therapeutically effective amount or dose” or“therapeutically sufficient amount or dose” or “effective or sufficientamount or dose” refer to a dose that produces therapeutic effects forwhich it is administered. The exact dose will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003,Gennaro, Ed., Lippincott, Williams & Wilkins). In sensitized cells, thetherapeutically effective dose can often be lower than the conventionaltherapeutically effective dose for non-sensitized cells.

As used herein, the term “vascular prosthesis” refers to a prosthesisfor the circulatory system of a mammal

II. Compounds of the Present Invention

Macrocyclic lactones, their salts, prodrugs, tautomers and isomers willbe referred to collectively as “macrocyclic lactones” in this invention.

The compounds of the present invention are macrocyclic lactone compoundsof the following formula:

wherein R¹, R², R³, R⁵, R⁶, R⁸, M¹, M², M³, M⁴, M⁵, M⁶ and M⁷ are eachindependently a member selected from the group consisting of H, C₁₋₆alkyl, OH and C₁₋₆ hydroxyalkyl; R⁴, R⁷ and R⁹ are each independentlyselected from the group consisting of C₁₋₆ alkoxy and OH;

R¹⁰ is a member selected from the group consisting of H, —OH, —OP(O)Me₂,

—O—(CH₂)_(n)—OH and —O—(CH₂)_(m)—O—(CH₂)_(o)—CH₃, wherein subscripts nand m are each independently from 2 to 8 and subscript o is from 1 to 6;each of L¹ and L⁴ are independently selected from the group consistingof:

wherein each M⁸ is independently a member selected from the groupconsisting of C₁₋₆ alkyl, OH and C₁₋₆ hydroxyalkyl; each of L² and L³are independently selected from the group consisting of:

with the proviso that when R¹, R⁶, R⁸, M⁶ and M⁷ are Me, R³, R⁵, M¹, M²,M³, M⁴ and M⁵ are H, R⁴, R⁷ and R⁹ are OMe, R¹⁰ and M⁸ are OH, L² and L³are —CH═CH—, and L¹ and L⁴ are

R² is other than OH;

with the proviso that when R¹, R⁶, R⁸, M⁶ and M⁷ are Me, R², R³, R⁵, M¹,M², M³, M⁴ and M⁵ are H, R⁷ and R⁹ are OMe, R¹⁰ and M⁸ are OH, L² and L³are —CH═CH—, and L¹ and L⁴ are

R⁴ is other than OH;

with the proviso that when R¹,R⁶, R⁸, M⁶ and M⁷ are Me, R², R³, R⁵, M¹,M², M³, M⁴ and M⁵ are H, R⁴ and R⁷ are OMe, R¹⁰ and M⁸ are OH, L² and L³are —CH═CH—, and L¹ and L⁴ are

R⁹ is other than OH;

with the proviso that when R¹, R⁶, R⁸, M⁶ and M⁷ are Me, R², R³, R⁵, M¹,M², M³, M⁴ and M⁵ are H, R⁴, R⁷ and R⁹ are OMe, M⁸ is OH, L² and L³ are—CH═CH—, and L¹ and L⁴ are

R¹⁰ is other than OH, —OP(O)Me₂,

—O—(CH₂)_(n)—OH and —O—(CH₂)_(m)—O—(CH₂)_(o)CH₃; and salts, hydrates,isomers, metabolites, N-oxides and prodrugs thereof.

In some embodiments, R¹⁰ is

In some other embodiments, R¹⁰ is

In other embodiments, R¹⁰ is —OP(O)Me₂.

In still other embodiments, R¹⁰ is

In another embodiment, R¹⁰ is

In a further embodiment, the compound is a compound of FIG. 2. In otherembodiments, at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ isOH. In still other embodiments, R⁴ is OH. In yet other embodiments, R¹,R⁶ and R⁸ are each methyl, R², R³ and R⁵ are each H, R⁴ and R¹⁰ are eachOH, R⁷ and R⁹ are each OMe, M¹, M², M³, M⁴ and M⁵ are each H, M⁶ and M⁷are each methyl, L¹ and L⁴ are each

and L² and L³ are each

The invention provides macrocyclic lactone compounds which are describedin detail with Formula IA, IB, IC and ID.

In one embodiment, the composition contains macrocyclic lactones whichinclude hydroxy, demethyl, hydroxydemethyl and epoxide macrocycliclactones.

The structure of certain macrocyclic lactones with some potential sitesfor chemical modifications to provide compounds of the present inventionis shown below.

wherein squares represent the demethylation positions; circles representthe hydroxylation positions; triangles represent the epoxidationpositions; curved lines represent the

N-oxidation position; dashed lines represent the position forring-opening position; R¹⁰ is a member selected from the groupconsisting of —OH,

—OP(O)Me₂, —R^(a)OH, where R^(a) is alkyl, such as —(CH₂)₂ to (CH₂)₇ and—R^(b)OR^(c), where R^(b) is C₂₋₆ alkylene and R^(c) is C₁₋₅ alkyl, suchas 40-O-(ethoxyethyl) rapamycin.

In some embodiments, the compounds of the present invention includecertain demethyl macrocyclic lactones, such as 16-O-demethyl macrocycliclactone, 39-O-demethyl macrocyclic lactone, 27-O-demethyl macrocycliclactone, 16, 27-bis-O-demethyl macrocyclic lactone, 27,39-bis-O-demethyl macrocyclic lactone, 16, 39-bis-O-demethyl macrocycliclactone, individually or in combination with each other, as shown inFormula IA.

wherein

Each of R⁴, R⁷ and R⁹ are selected from the group of consisting of —OCH₃and —OH.

R⁴ and R⁷ are both, independently, selected from the group of consistingof —OH and —OCH₃.

R⁷ and R⁹ are both, independently, selected from the group of consistingof —OH and —OCH₃.

R⁴ and R⁹ are both, independently, selected from the group of consistingof —OH and —OCH₃.

R⁴, R⁷ and R⁹ are each, independently, selected from the group ofconsisting of —OH and —OCH₃.

And wherein R¹⁰ is as described above.

In another embodiment, the compounds of the present invention includescompositions of hydroxyl macrocyclic lactone, such as 11-hydroxylmacrocyclic lactone, 12-hydroxyl macrocyclic lactone, 14-hydroxylmacrocyclic lactone, 24-hydroxyl macrocyclic lactone, 25-hydroxylmacrocyclic lactone, 25-methyl alcohol macrocyclic lactone, 31-methylalcohol macrocyclic lactone,35-methyl alcohol macrocyclic lactone,13-methyl alcohol macrocyclic lactone, individually or in combinationwith each other, as shown in Formula IB.

wherein

Each of R¹, R⁶, R⁸, M⁶, M⁷, M⁸ and M^(8a) are selected from the group ofconsisting of —CH₃, —CH₂OH and —OH. Each of R², R³, R⁵, M¹, M², M³, M⁴and M⁵ are selected from the group of consisting of —H and —OH. R⁹ isselected from the group of consisting of —OH and —OCH₃. L₂ is selectedfrom the group of consisting of

And wherein R¹⁰ is as described above.

In another embodiment, the compounds of the present invention includecompositions of epoxide macrocyclic lactone, such as 19, 20-21, 22-29,30 tris epoxide macrocyclic lactone, 17, 18-19, 20-21, 22 tris epoxidemacrocyclic lactone and 17, 18-29, 30 bis epoxide macrocyclic lactone,as shown in Formula IC.

And wherein R¹⁰ is as described above.

Formula ID

In another embodiment, the compounds of the present invention includecompositions of N-oxidized macrocyclic lactone, 11-hydroxyl with 10,14ring opening macrocyclic lactone, seco-macrocyclic lactone (with 1, 34ring opened), as shown in Formula ID.

Wherein each of R¹, R⁶, R⁸, M⁶, M⁷, M⁸ and M^(8a) are independentlyselected from the group of consisting of —CH₃, —OH and C₁₋₆hydroxyalkyl. Each of R², R³, R⁵, M¹, M², M³, M⁴ and M⁵ areindependently selected from the group of consisting of —H and —OH. R⁴,R⁷ , R⁹ are independently selected from the group of consisting of —OHand —OCH₃. L² is selected from the group of consisting of Error! Objectscannot be created from editing field codes. and Error! Objects cannot becreated from editing field codes.

In another embodiment, the compounds of the present invention are acombination of Formula IA and Formula IB, including composition ofdemethylhydroxy macrocyclic lactone such as 14-hydroxy-39-O-demethylmacrocyclic lactone, 16,39-bis-O-demethyl-24-hydroxy macrocycliclactone, 16,27-bis-O-demethyl-24-hydroxy macrocyclic lactone,27,39-bis-O-demethyl-24-hydroxy macrocyclic lactone individually or incombination with each other.

In another embodiment, the compounds of the present invention are acombination of Formula IA and Formula IC, including composition ofepoxide demethyl macrocyclic lactone such as17,18-19,20-bis-epoxide-16-O-demethyl macrocyclic lactone,17,18-29,30-bis-epoxide-16-O -demethyl macrocyclic lactone, individuallyor in combination with each other.

In yet another embodiment, the compounds of the present invention are acombination of Formula IA, Formula IB and Formula IC, includingcomposition of epoxide demethylhydroxyl macrocyclic lactone such as

17,18-19,20-bis-epoxide-16-O-demethyl-24-hydroxy-macrocyclic lactone,17,18-29,30-bis-epoxide- 16-O-demethyl-24-hydroxy- macrocyclic lactone,individually or in combination with each other.

This invention also covers the compositions of salts, hydrates, isomers,tautomers, metabolites, N-oxides and prodrugs of compounds of thepresent invention of Formula IA, IB, IC and ID.

Structures of preferred embodiments (A,B,C, . . . AA,AB,AC, . . .FB,FC,FD) amongst compounds of the present invention of Formula IA, IB,IC and ID, and their combinations, are shown in Table 1, along with somein FIG. 2A-2FV.

include both sterocenter:

and

TABLE 1 Table of preferred compounds.

* R¹ Me R² H R³ H R⁴ OMe R⁵ H R⁶ Me R⁷ OMe R⁸ Me R⁹ OMe R¹⁰ OH M¹ H M² HM³ H M⁴ H M⁵ H M⁶ Me M⁷ Me L¹  

L²  

L³  

L⁴  

A OH B OH C OH D OH E OH F OH G OH H

I OH

J OH

K OH

L OH

M OH

N OH

O OH

P OH

Q OH

R OH

S OH

T OH

U OH

V OH

W OH —OP(O)Me₂ X OH —OP(O)Me₂ Y OH —OP(O)Me₂ Z OH —OP(O)Me₂ AA OH—OP(O)Me₂ AB OH —OP(O)Me₂ AC OH —OP(O)Me₂ AD OH OH AE OH

AF OH

AG OH

AH OH

AI OH

AJ

AK OH OH

AL OH H—(CH₂CH₂O)₂— AM OH H—(CH₂CH₂O)₂— AN OH H—(CH₂CH₂O)₂— AO OHH—(CH₂CH₂O)₂— AP OH H—(CH₂CH₂O)₂— AQ OH H—(CH₂CH₂O)₂— AR OH AS

AT OH

AU OH

AV OH

AW OH OH

AX OH OH

AY

AZ

BA OH BB OH BC OH BD OH BE OH BF CH₂OH BG

BH CH₂OH BI CH₂OH BJ

BK CH₂OH BL CH₂OH BM

OH BN

OH BO

OH BP

OH BQ

OH BR CH₂OH

BS

BT

CH₂OH BU CH₂OH

BV

BW

CH₂OH BX CH₂OH

BY

OH BZ

OH CA

OH CB

OH CC

OH CD CH₂OH

CE

CF

CH₂OH CG CH₂OH

CH

CI

CH₂OH CJ CH₂OH

CK —OP(O)ME₂ OH CL —OP(O)ME₂ OH CM —OP(O)ME₂ OH CN —OP(O)ME₂ OH CO—OP(O)ME₂ OH CP CH₂OH —OP(O)ME₂ CQ —OP(O)ME₂

CR —OP(O)ME₂ CH₂OH CS CH₂OH —OP(O)ME₂ CT —OP(O)ME₂

CU —OP(O)ME₂ CH₂OH CV CH₂OH —OP(O)ME₂ CW

OH CX

OH CY

OH CZ

OH DA

OH DB CH₂OH

DC

DD

CH₂OH DE CH₂OH

DF

DG

CH₂OH DH CH₂OH

DI

OH DJ

OH DK

OH DL

OH DM

OH DN CH₂OH

DO

DP

CH₂OH DQ CH₂OH

DR

DS

CH₂OH DT CH₂OH

DU

DV OH

DW OH

DX OH

DY OH OH

DZ

EA OH

EB OH

EC OH

ED OH OH

EE

EF

EG OH

EH OH

EI OH

EJ OH OH

EK

EL —OP(O)ME₂

EM OH —OP(O)ME₂

EN OH —OP(O)ME₂

EO OH —OP(O)ME₂

EP OH OH —OP(O)ME₂

EQ —OP(O)ME₂

ER

ES OH

ET OH

EU OH

EV OH OH

EW

EX OH

EY OH

EZ OH

FA OH OH

FB

FC —OP(O)ME₂

FD

*Rapamycin. All —OH substitutions represent a mixture of both the R andthe S isomer.

In some embodiments, the compound of the present invention has thefollowing structure:

This invention covers compounds in which the stereochemistry of the16-position is racemic (R,S) as well as the individual R and Sstereoisomers at the 16-position and all other isomers of the compound.

The invention covers compounds with different polymorphic forms. Thisincludes 16-O-demethyl macrocyclic lactone having different polymorphicforms. For example, different polymorphs form of 16-O-demethylmacrocyclic lactone are obtained from using dichloromethylene and fromusing a mixture of methanol and water

There have been different numbering schemes proposed for macrocycliclactones. To avoid confusion, when specific macrocyclic lactones arenamed herein, the names are given with reference to macrocyclic lactoneusing the numbering scheme of the above chemical formula. This inventionalso covers all the macrocyclic lactones which have different name dueto a different numbering scheme if the same functional group exists inthe same location within the chemical structure. For example,39-O-demethyl macrocyclic lactone is the same compound as 41-O-demethylmacrocyclic lactone and 16-O-demethyl macrocyclic lactone is the samecompound as 7-O-demethyl macrocyclic lactone.

The compounds of the present invention can be prepared by a variety ofmethods. In some embodiments, the compounds of the present invention aresynthesized biologically by genetically modifying the strains oforganisms to produce the compounds of the present invention or by othermeans.

In another embodiment the compounds of the present invention areprepared using chemical synthesis. Chemical synthesis of the compoundsof the present invention can utilize the 17-18, 19-20, 21-22 trienestructure in macrocyclic lactones, which facilitates the acid catalyzednucleophilic substitution of the C16 methoxy group and allows theintroduction of a number of different substitutions and allows theselective manipulation of macrocyclic lactone effector domain. The C16methoxy group in macrocyclic lactones is manipulated towards acidicreagents to produce the compounds of the present invention. For example,replacement of C16 methoxy groups with different nucleophiles such asalcohols, thiols and electron rich aromatic groups can be accomplished.This method of synthesis can be performed without protection anddeprotection steps.

The method of synthesis of using acidic reagents with compounds havingtriene functional groups can be applied to other compounds having trienefunctional groups to synthesize corresponding compound analoguesproviding a method of synthesis without protection and deprotectionsteps

In some embodiments, the present invention provides a method of making acompound of the present invention, the method comprising contacting amacrocyclic lactone with an acid to replace an alkoxy group with anucleophile, thereby making a compound of the present invention. In someother embodiments, the macrocyclic lactone is rapamycin. In otherembodiments, the nucleophile is a member selected from the groupconsisting of —OH, —SH and electron rich aromatic groups. One of skillin the art will appreciate that other methods are useful for preparingthe compounds of the present invention.

Synthesis of demethyl macrocyclic lactone is provided in FIG. 14 using16-O-demethyl macrocyclic lactone as an example. Synthesis of hydroxymacrocyclic lactone is provided in FIG. 15 using 19, 20 bis-hydroxymacrocyclic lactone as an example. Synthesis of epoxide macrocycliclactone is provided in FIG. 16 using 17,18-29,30-bis epoxide macrocycliclactone as an example.

In another embodiment, the present invention provides a method of makinga compound of the present invention, the method comprising contacting amacrocyclic lactone with a suitable agent such as a peracid or peroxide,to modify an alkene group to an epoxide, thereby making a compound ofthe present invention. Peracids useful in the methods of the presentinvention include, but are not limited to, peroxy-carboxylic acids ofthe formula R—C(O)—OOH, where the R group can be groups such as H,alkyl, alkene or aryl. In some embodiments, the peracid can beperoxy-acetic acid or meta-chloro-peroxybenzoic acid (MCPBA). Peroxidesuseful in the methods of the present invention include, but are notlimited to, hydrogen peroxide. One of skill in the art will appreciatethat other epoxidation reagents are useful in the present invention.

The compounds of the present invention are optionally deuterated.

In some embodiments, the present invention provides compounds of thepresent invention which have a similar potency to the correspondingparent macrocyclic lactone. In another embodiment, the present inventionprovides compounds of the present invention which are less potent thanthe corresponding parent macrocyclic lactone in order to improve thesafety profile of the compounds.

III. Delivery of the Compounds of the Present Invention

The compounds of the present invention can be administered in anyappropriate manner. In some embodiments, the compounds are administeredorally, intramuscularly, intraperitoneally, subcutaneously, pulmonarily,mucosally, transdermally, intravascularly, intraocularly orintravitreally through the eye, and others. In other embodiments, thecompounds are administered site specifically through temporary orpermanent drug delivery means such as an implant or a combination ofsystemic and site specific means. Examples include, but are not limitedto catheter, stent, vascular wrap, pump, shunt or other temporary orpermanent drug delivery means.

A. Device

In some embodiments, the present invention provides a device forintracorporeal use, the device comprising a vascular prosthesis; and atleast one source of a compound of the present invention.

In other embodiments, the present invention provides a device configuredto release the compound to a body lumen or organ within anintracorporeal body to inhibit cell proliferation or cell migration. Ina further embodiment, the device is configured to release the compoundto a body lumen or organ within an intracorporeal body to inhibit smoothmuscle cell proliferation or neo-vascularization.

In another embodiment of the present invention the drug delivery meansis a device such as an implant including graft implants, vascularimplants, non-vascular implants, implantable luminal prostheses, woundclosure implants, drug delivery implants, sutures, biologic deliveryimplants, urinary tract implants, inter-uterine implants, organimplants, ophthalmic implants, bone implants including bone plates, bonescrews, dental implants, spinal disks, or the like.

When the device is configured for treatment of ophthalmic conditions ordiseases, the implant of the present invention can be implantedintraocularly or intravitreally by an intervention procedure. Suchimplants can be non-biodegradable, biodegradable, removable orpermanent. In other embodiments, implants can be placed in the duct,such as the tear duct. In still other embodiments, implants can beplaced adjacent to the ocular body, or intraocularly, adjacent to thevitreal body or intravitreally. One of skill in the art will appreciatethat other locations are useful in the present invention.

The implant typically allows for one or more of the following: support,contain, hold together, affix, plug, close, maintain, deliver drug,deliver biologics—to a body lumen, organ, vessel, conduit, muscle,tissue mass or bone for the prevention or treatment of diseaseconditions, such as for example hyper-proliferative diseases,restenosis, cardiovascular disease, inflammation, wound healing, cancer,aneurysm, diabetic disease, abdominal aortic aneurysm, hyper-calcemia,or others.

The implant of the present invention can be formed of metal, metalalloy, polymer, ceramic, semi-metal, nanocomposites or combinationthereof. For example, an implant can be made from metal such astantalum, iron, magnesium, molybdenum or others; from a degradable ornon degradable metal alloy such as 316L stainless steel, carbon steel,magnesium alloy, NI—Ti, Co—Cr such as L605, MP35 or other; from apolymer that is degradable or non-degradable such as poly lactic acid,poly glycolic acid, poly esters, polyamide, copolymers or others orblends of polymers; combination of metals and metals or metal alloyssuch as implant made from combination of layers of stainless steel andtantalum or others; nanocomposites such as nano carbon fibers or nanocarbon tubules or others.

In another embodiment, the present invention provides a device whereinthe implant is a vascular prosthesis. In some embodiments, the vascularprosthesis comprises an expandable structure. In other embodiments, thevascular prosthesis comprises a stent, graft, or a scaffold formed atleast in part from an open lattice. In still other embodiments, thevascular prosthesis is a stent.

In another embodiment, the compounds of the present invention can beapplied adjacent to the surface of the implant. For example thecompounds of the present invention can be incorporated within theimplant, contained within a coating, or carried on the implant.

In some embodiments, the present invention provides a device comprisinga vascular prosthesis wherein the vascular prosthesis has a luminal anda tissue facing surface, and wherein the compound is associated with atleast one of the luminal or tissue facing surfaces.

In a further embodiment, the compounds of the present invention areapplied on all implant surfaces. In another embodiment, the compounds ofthe present invention are applied only to the abluminal or luminalsurface. In yet another embodiment, the compounds of the presentinvention are applied only to high stress or low stress areas.

In another embodiment, the compounds of the present invention arecontained within an erodible or non-erodible filament or filaments thatare adjacent to the implant.

An example of a stent configuration for carrying a compound of thepresent invention is illustrated in FIG. 3 in a contracted state. Thestent body is formed of multiple rings 110. The rings are formed ofcrowns 120 and struts 130 in a generally expandable undulatingconfigurations such as, zigzag, sawtooth, sinusoidal wave or other. Thebody is joined by links or connectors 140. It is understood that theconnectors may be of any length or shape, or may not be needed if thecrowns are directly attached to each other. The stent has a typicalcontracted state diameter of between 0.25-4 mm, or more preferablybetween 0.7 to 1.5 mm, and a length of between 5 and 100 mm. In itsexpanded state, the stent diameter is typically at least twice and up to10 times or more than that of the stent in its contracted state. Thus, astent with a contracted diameter of between 0.7 to 1.5 mm may expandradially to 2 to 10mm or more.

Drug eluting stents with potent macrocyclic lactone compounds such asrapamycin (Cypher™) have resulted in late lumen loss in the range ofapproximately 0.01 mm to 0.2 mm at approximately 4 months to 12 monthsangiographic follow up. The late lumen loss with bare metal stents haveranged from approximately 0.70 mm to 1.2 mm for the same time period.Lower late lumen loss typically decreased the percent stenosis. However,significantly lower late lumen loss with drug eluting stents as comparedto bare metal stents in some cases results in inadequate tissue coverageof the stent surface which potentially may increase incidence of latestent thrombosis.

In a preferred embodiment, the late lumen loss for stent carryingcompounds of the present invention after approximately 4 to 12 monthsafter implantation is greater than the late lumen loss for stentcarrying the corresponding parent macrocyclic lactone by 0.05 mm to 0.6mm, preferably by 0.1 mm to 0.4 mm, more preferably by 0.15 to 0.3 mm.For example, late lumen loss after implantation ranges from 0.01 mm to0.6 mm, preferably from 0.1 mm to 0.5 mm and most preferably from 0.2 mmto 0.4 mm. In another embodiment, the present invention provides latelumen loss for stent carrying compounds of the present invention similarto a stent carrying the corresponding parent macrocyclic lactone. Inanother preferred embodiment, the present invention provides late lumenloss for stent carrying compounds of the present invention being higherthan for a stent carrying the corresponding parent macrocyclic lactone.Higher late lumen can provide increased tissue coverage of the stentwhich can improve safety of the stent.

In another preferred embodiment, the percent stenosis approximately 4 to12 months after implantation for a stent carrying compounds of thepresent invention is greater by 1 to 30 percentage than the percentstenosis for a stent carrying the corresponding parent macrocycliclactone, preferably from 3 to 20 percentage, more preferably from 5 to15 percentage. In yet another preferred embodiment, the presentinvention provides percent stenosis for a stent carrying compounds ofthe present invention similar to a stent carrying the correspondingparent macrocyclic lactone. In another preferred embodiment, the presentinvention provides percent stenosis for a stent carrying compounds ofthe present invention higher than a stent carrying the correspondingparent macrocyclic lactone. In another preferred embodiment, the percentstenosis for a stent carrying compounds of the present invention ishigher than a stent carrying the corresponding parent macrocycliclactone but lower than the bare metal stent. Higher stenosis can provideincreased tissue coverage of the stent which can improve safety of thestent.

In some embodiments, the present invention provides a device wherein theamount of compounds of the present invention on the implant is less thanabout 1 g/cm². In other embodiments, the amount of compounds on theimplant can range from about 1 nanogram/cm² to about 1000 microgram/cm²,preferably from about 1 microgram/cm² to about 500 microgram/cm², morepreferably from about 10 microgram/cm² to about 400 microgram/cm². Instill other embodiments, the amount of compound on the implant is lessthan about 1 mg. In yet other embodiments, the amount of compound on theimplant is from about 1 μg to about 50 mg, preferably from about 100 μgto about 10 mg, more preferably from about 200 μg to about 500 μg.

In a further embodiment, the present invention provides a device whereinthe concentration of the compound of the present invention in the tissueadjacent to the implant is from about 0.001 ng/gm tissue to about 1000μg/gm tissue, preferably from about 1 ng/gm tissue to about 500 μg/gmtissue, more preferably from about 100 ng/gm tissue to about 100 μg/gmtissue.

In another embodiment, the compounds of the present invention can bereleased from the implant over a period ranging from less than 5 minutesto 2 years, preferably from 3 days to 6 months, more preferably from 1week to 3 months. In other embodiments, the compounds of the presentinvention can be released from the implant over a period greater than 1day, preferably greater than 2 weeks, more preferably greater than 1month. In another embodiment, the compounds of the present invention canrequire greater than 2 years to be fully released from the stent. Insome embodiments, the amount of compound released over the given timeperiod is at least 25%. In other embodiments, the amount of compoundreleased is at least 50%. In still other embodiments, the amount ofcompound released is at least 75%. In yet other embodiments, the amountof compound released can be at least 80, 85, 90, 91, 92, 93, 94, 95, 96,97, 98 or 99%.

In a further embodiment, the present invention provides a device whereinat least 75% of the compound is released from the device in a periodfrom about 1 day to about 2 years. In another embodiment, at least 90%of the compound is released from the device in a period from about 3 dayto about 6 months. In still another embodiment, at least 90% of thecompound is released from the device in a period from about 1 week toabout 3 months.

When the compounds of the present invention are administered viainjection or eye drop through an intraocular or intravitreal body, theconcentration of compound alone or within the polymer matrix, solvent orcarrier can vary from 1 μg/ml to 5 mg/ml, and preferably from 5 μg/ml to30 μg/ml. Following administration, the concentration of the compound ofthe present invention in the tissue adjacent to the site ofadministration can be from about 0.1 nM to 500 μM, preferably from about1 nM to 100 μM, more preferably from about 10 nM to 10 μM. One of skillin the art will appreciate that other concentrations of the compounds ofthe present invention are useful.

The compounds of the present invention can be released from the implantvia any means known in the art. In some embodiments, the implantreleases the compound through active or passive means. In otherembodiments, the implant releases the compound through osmotic pressureor diffusion. One of skill in the art will appreciate that other meansof releasing the compound from the implant are useful in the presentinvention.

In some embodiments, the present invention provides a device thatfurther includes a therapeutic agent, such as those described below. Insome other embodiments, the therapeutic agent is released prior to,concurrent with, or subsequent to the release of the compound. In otherembodiments, the compound is released from a first source and thetherapeutic agent is released from a second source. In still otherembodiments, the compound and the therapeutic agent are released from asingle source.

B. Administration

The compounds of the present invention can be administered systemicallyon a daily, intermittent or one-time dose basis. The daily systemic dosecan range from 0.1 mg to 20 mg preferably 0.5 mg to 10 mg, mostpreferably from 1 mg to 5 mg per day. One of skill in the art willappreciate that other doses are also useful in the present invention.

The compounds of the present invention can be released from the implantat rates ranging from about 1 nanogram/cm²/day to about 1000microgram/cm²/day, preferably from about 1 microgram/cm²/day to about200 microgram/cm²/day, more preferably from about 5 microgram/cm²/day toabout 100 microgram/cm²/day.

When the device of the present invention is configured for treatment ofophthalmic conditions or diseases, the compounds of the presentinvention can be administered through the eye as an eye drop or aninjection on a daily, intermittent or one time dose basis. The dose canrange from 0.1 μg to 30 mg, preferably from 10 μg to 10 mg, mostpreferably from 100 μg to 1 mg per day. One of skill in the art willappreciate that other doses are also useful in the present invention.

C. Pharmaceutical Formulations

In some embodiments, the present invention provides a pharmaceuticalcomposition comprising a pharmaceutically acceptable excipient and acompound of the formula:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, M¹, M², M³, M⁴, M⁵, M⁶, M⁷, L¹,L², L³ and L⁴ are as described above; and salts, hydrates, isomers,metabolites, N-oxides and prodrugs thereof.

In some other embodiments, the present invention provides apharmaceutical composition including a pharmaceutically acceptableexcipient and a compound of the formula:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, M¹, M², M³, M⁴, M⁵, M⁶,M⁷, L¹, L², L³ and L⁴ are as described above; and salts, hydrates,isomers, metabolites, N-oxides and prodrugs thereof.

In still other embodiments, the present invention provides apharmaceutical composition including a pharmaceutically acceptableexcipient and a compound of the formula:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, M¹, M², M³, M⁴, M⁵, M⁶,M⁷, L¹, L², L³ and L⁴ are as described above; and salts, hydrates,isomers, metabolites, N-oxides and prodrugs thereof.

In another embodiment, the pharmaceutical composition includes acompound of FIG. 2. In other embodiments, R¹, R⁶ and R⁸ are each methyl,R², R³ and R⁵ are each H, R⁴ and R¹⁰ are each OH, R⁷ and R⁹ are eachOMe, M¹, M², M³, M⁴ and M⁵ are each H, M⁶ and M⁷ are each methyl, L¹ andL⁴ are each

and L² and L³ are each

In some embodiments, the present invention provides a pharmaceuticalcomposition wherein the pharmaceutically acceptable excipient is amember selected from the group consisting of a polymer, a solvent, anantioxidant, a binder, a filler, a disintegrant, a lubricant, a coating,a sweetener, a flavor, a stabilizer, a colorant, a metal, a ceramic anda semi-metal. In other embodiments, the pharmaceutically acceptableexcipient is a polymer.

The active ingredients of the present invention may be mixed withpharmaceutically acceptable carriers, diluents, adjuvants, excipients,or vehicles, such as preserving agents, fillers, polymers,disintegrating agents, glidants, wetting agents, emulsifying agents,suspending agents, sweetening agents, flavoring agents, perfumingagents, lubricating agents, acidifying agents, and dispensing agents,depending on the nature of the mode of administration and dosage forms.Such ingredients, including pharmaceutically acceptable carriers andexcipients that may be used to formulate oral dosage forms, aredescribed in the Handbook of Pharmaceutical Excipients, AmericanPharmaceutical Association (1986), incorporated herein by reference inits entirety. Examples of pharmaceutically acceptable carriers includewater, ethanol, polyols, vegetable oils, fats, waxes polymers, includinggel forming and non-gel forming polymers, and suitable mixtures thereof.Examples of excipients include starch, pregelatinized starch, Avicel,lactose, milk sugar, sodium citrate, calcium carbonate, dicalciumphosphate, and lake blend. Examples of disintegrating agents includestarch, alginic acids, and certain complex silicates. Examples oflubricants include magnesium stearate, sodium lauryl sulphate, talc, aswell as high molecular weight polyethylene glycols. One of skill in theart will appreciate that other different excipients can be used informulations according to the present invention and the list providedherein is not exhaustive.

Suitable nondegradable or slow degrading polymer coatings include, butare not limited to, polyacrylamide, poly-N-vinylpyrrollidone,polydimethyl acrylamide, polymers and copolymers of2-acrylamido-2-methyl-propanesulfonic acid, acrylic acid and methacrylicacid, polyurethane, polyethylenes imine, ethylene vinyl alcoholcopolymer, silicone, C-flex, nylons, polyamide, polyimide,polytetrafluoroethylene (PTFE), parylene, parylast, poly (methylmethacrylate), poly(n-butyl methacrylate), poly (butyl methacrylate)copolymer or blended with poly(ethylene vinyl acetate), poly(methylmethacrylate), poly (2-hydroxy ethyl methacrylate), poly(ethylene glycolmethacrylates), poly(vinyl chloride), poly(dimethyl siloxane),poly(ethylene vinyl acetate), polycarbonate, polyacrylamide gels, andthe like, including other synthetic or natural polymeric substances;mixtures, copolymers, or combinations thereof.

Suitable biodegradable polymer coatings include, but are not limited to,poly(lactic acid), polylactates, poly(glycolic acid), polyglycolates andcopolymers, poly dioxanone, poly(ethyl glutamate),poly(hydroxybutyrate), polyhydroxyvalerate and copolymers,polycaprolactone, polyanhydride, poly(ortho esters); poly(ether esters),poly ethylene glycols, poly(ethylene oxide), poly (trimethyl carbonate),polyethylenecarbonate, copolymers of poly(ethylenecarbonate) andpoly(trimethyl carbonate), poly(propylene carbonate),poly(iminocarbonates), starch based polymers, cellulose acetatebutyrate, polyester amides, polyester amines, polycyanoacrylates,polyphosphazenes, Poly N-vinyl-2-pyrrolidone, poly maleic anhydride,hyaluronic acid (hyaluronate), chondroitin sulfate, dermatan sulfate,carboxymethylcellulose, heparin sulfate, keratan sulfate,carboxymethylhydroxypropylcellulose, carboxymethylhydroxethylcellulose,cellulose sulfate, cellulose phosphate, carboxymethylguar,carboxymethylhydroxypropylguar, carboxymethylhydroxyethylguar, xanthangum, carrageenan, anionic polysaccharides, anionic proteins andpolypeptides, quaternary ammonium compounds including stearyl ammoniumchloride and benzyl ammonium chloride, copolymers and other aliphaticpolyesters, or suitable copolymers thereof including copolymers ofpoly(L-lactic acid) and poly(e-caprolactone); mixtures, copolymers,ionic polymers, or combinations thereof.

Suitable natural coatings include: fibrin, albumin, collagen, gelatin,glycosoaminoglycans, oligosaccharides and poly saccharides, chondroitin,chondroitin sulphates, hypoxyapatite, phospholipids, phosphorylcholine,glycolipids, fatty acids, proteins, cellulose, and mixtures, copolymers,or combinations thereof.

Suitable non polymeric coatings include metallic coatings such astungsten, magnesium, cobalt, zinc, iron, bismuth, tantalum, gold,platinum, stainless steel such as 316L, 304, titanium alloys; ceramicscoatings such as silicon oxide; semi-metals such as carbon, nanoporouscoatings; or combination thereof.

In some embodiments, the pharmaceutically acceptable excipient is apolymer is selected from the group consisting of polyurethane,polyethylene imine, ethylene vinyl alcohol copolymer, silicone, C-flex,nylons, polyamide, polyimide, polytetrafluoroethylene (PTFE), parylene,parylast, poly(methacrylate), poly(vinyl chloride), poly(dimethylsiloxane), poly(ethylene vinyl acetate), polycarbonate, polyacrylamidegels, poly (methyl methacrylate), poly(n-butyl methacrylate), poly(butyl methacrylate) copolymer or blended with poly(ethylene vinylacetate), poly(methyl methacrylate), poly (2-hydroxy ethylmethacrylate), poly(ethylene glycol methacrylates), poly(ethylenecarbonate), Poly L lactide-glycolide copolymer, poly Llactide-trimethylene carbonate copolymer and Poly L-lactide. In afurther embodiment, the polymer is poly(n-butylmethacrylate).

In a further embodiment, the present invention provides a compositionwherein the compound is present in an amount of at least 10% (w/w) in amixture of the compound and the polymer. In another embodiment, thecompound is present in an amount of at least 20, 25, 30, 40, 50, 55, 60,70, 75, 80 and 90% (w/w). In other embodiments, the compound is presentin an amount of at least 25% (w/w). In some other embodiments, thecompound is present in an amount of at least 50% (w/w). In still otherembodiments, the compound is present in an amount of at least 75% (w/w).One of skill in the art will appreciate that other compositions areuseful in the present invention.

In another embodiment, the compounds of the present invention can beapplied onto a stent without a coating. In another embodiment, compoundsof the present invention can be applied onto a stent in combination witha polymer coating such as a compounds of the present invention-polymermatrix. The compounds of the present invention can be fully or partiallycrystallized or in an amorphous form. The polymer can be non degradable,partially degradable or fully degradable. The coating can also be anon-polymeric such as metallic coating. In another embodiment, thecompounds of the present invention can be applied on a stent alone orcontained in a coating with a polymer or non polymer topcoat. In anotherembodiment, the stent includes an underlayer coating disposed betweenthe stent surface and the compounds of the present invention orcompounds of the present invention -polymer matrix. Suitable underlayercoatings can be polymeric such as paralyne C, parylene N, ethylene vinylalcohol (EVOH), polycaprolactone, ethylvinyl hydroxylated acetate (EVA),or others or combination thereof or non polymeric such as metallic orceramic or others.

The coatings can be applied by any of the different methods whichinclude but are not limited to spraying, ultrasonic deposition, dipping,inkjet dispension, plasma deposition, ion implantation, sputtering,evaporation, vapor deposition, pyrolysis, electroplating, glow dischargecoating, or others or combination thereof.

The coating thickness can range from 1 nanometer to 100 micrometers,preferably from 100 nanometers to 50 micrometers, more preferably from 1micrometer to 20 micrometers.

The compounds of the present invention can be combined with antioxidantsor stabilizers to prevent degradation due to oxidation or other means.Antioxidants include but are not limited to butylated hydroxytoluene(BHT), ferrous sulfate, ethylenediamine-tetra-acetic acid (EDTA), orothers. Stabilizers include, but are not limited to, amglene,hydroquinone, quinine, sodium metabisulfite or others. Antioxidants andstabilizers can be combined with the compounds directly or blended withthe compound formulation such as compound-polymer matrix to reduceconformation change or degradation during manufacturing processes andincrease shelf life or storage life of the compounds or compoundcontaining implant. The amount of antioxidants such as BHT in thecompounds can range from 0.01% to 10%, preferable from 0.05% to 5% andmost preferable from 0.1% to 1%. The amount of stabilizers such asamylene in the compounds can range from 0.001% to 0.1%, preferably from0.005% to 0.05%, most preferably from 0.01% to 0.02%. One of skill inthe art will appreciate that other antioxidants and stabilizers areuseful in the present invention.

The compounds of the present invention can be administered incombination with a therapeutic agent such as anti-platelet,anti-thrombotic, anti-inflammatory, anti-angiogenic, anti-proliferative,immunosuppressant, anti-cancer or other agents or combinations thereof.One of skill in the art will appreciate that other therapeutic agentsare useful in the present invention.

The therapeutic agents can be incorporated on the stent together withthe compounds of the present invention and/or separately from compoundsof the present invention. At least a portion of the therapeutic agentcan be released from the stent prior to, concurrently or post release ofthe compounds of the present invention from the stent. The therapeuticagent can also be given separately through systemically or site specificadministration prior to, during or post delivery of compounds of thepresent invention.

For example, compounds of the present invention are given with antiplatelets or anti-thrombotics such as heparin, clopidogrel, coumadin,aspirin, ticlid or others. In another example, compounds of the presentinvention are given with anti-inflammatory agents such as aspirin,diclofenac, indomethacin, sulindac, ketoprofen, flurbiprofen, ibuprofen,naproxen, piroxicam, tenoxicam, tolmetin, ketorolac, oxaprosin,mefenamic acid, fenoprofen, nambumetone (relafen), acetaminophen, andmixtures thereof; COX-2 inhibitors, such as nimesulide, NS-398,flosulid, L-745337, celecoxib, rofecoxib, SC-57666, DuP-697, parecoxibsodium, JTE-522, valdecoxib, SC-58125, etoricoxib, RS-57067, L-748780,L-761066, APHS, etodolac, meloxicam, S-2474, tacrolimus, and mixturesthereof; glucocorticoids, such as hydrocortisone, cortisone, prednisone,prednisolone, methylprednisolone, meprednisone, triamcinolone,paramethasone, fluprednisolone, betamethasone, dexamethasone,fludrocortisone, desoxycorticosterone, or others or analogues of theabove or combinations thereof.

In some embodiments, the compounds of the present invention are combinedwith at least one therapeutic agent for treatment of an ophthalmiccondition or disorder. Any suitable therapeutic agent known to one ofskill in the art can be combined with the compounds of the presentinvention for use in the treatment of ophthalmic conditions or diseases.Therapeutic agents that can be combined with the compounds of thepresent invention include, but are not limited to, lucentis, avastin,macugan, volociximab, olopatadine, mydriatcs, dexamethasone,pilocarpine, tropicamide, quinolone, galentamine, fluocinoloneacetonide, triamcinolone acetonide, atropine, atropine sulfate, atropinehydrochloride, atropine methylbromide, atropine methylnitrate, atropinehyperduric, atropine N-oxide, phenylephrine, phenylephrinehydrochloride, hydroxyamphetamine, hydroxyamphetamine hydrobromide,hydroxyamphetamine hydrochloride, hydroxyamphetamine iodide,cyclopentolate, cyclopentolate hydrochloride, homatropine, homatropinehydrobromide, homatropine hydrochloride, homatropine methylbromide,scopolamine, scopolamine hydrobromide, scopolamine hydrochloride,scopolamine methylbromide, scopolamine methylnitrate, scopolamineN-oxide, tropicamide, tropicamide hydrobromide, tropicamidehydrochloride, pilocarpine, isopilocarpine, physostigmine, andquaternary ammonium compounds including stearyl ammonium chloride andbenzyl ammonium chloride, including mixtures, ionic salt s, andcombinations thereof.

Formulations of the compounds of the present invention for ophthalmicuses can include any polymer described above. The polymers useful insuch formulations can be of any size. In some embodiments, the polymerscan have a molecular weight of between about 50 kilo Daltons (kD) and8,000 kD. One of skill in the art will appreciate that polymers of othersizes are useful in the present invention.

In some embodiments, the compounds of the present invention can beadministrated alone or as part of compound-polymer formulation,compound-solvent formulation or compound-carrier formulation. Allformulations of the present invention may include active and inactiveingredients. Active ingredients include, but are not limited to,anti-inflammatory agents, immunomodulating agent and anti-infectiveagents, antioxidants, antibody, antibiotics, anti-angiogenics,anti-vascular endotherlial growth factor agent, antihistamines andlubricant. Inactive ingredients include, but are not limited to,carrier, solvent, inorganic materials, pH-adjustor, radio-opaque,radioactive, fluorescent, NMR contrast or other “reporter or indicator”materials. Examples of solvent in the compound-solvent formulationinclude, but are not limited to, water, saline, alcohol, and dimethylsulfoxide. Examples of carrier in the compound-carrier formulation areglycerin, paraffin, beeswax, ethylene glycol, propylene glycol,polyethylene glycol, and macrogels. Examples of inorganic materialsinclude, but are not limited to, boric acid, calcium chloride, magnesiumchloride, potassium chloride, sodium chloride, zinc chloride, sodiumborate, povidone, and dibasic sodium phosphate. Examples of pH-adjustorinclude, but are not limited to, sodium hydroxide, hydrogen chloride,buffer, and other inorganic and organic acid/base. Examples ofpreservative include, but are not limited to, benzalkonium chloride, anda polyquaternium. Examples of lubricant include, but are not limited to,carboxymethylcellulose sodium, polyethylene glycol, propylene glycol andethylene glycol. One of skill in the art will appreciate that otheractive and inactive ingredients, as well as solvents and carriers areuseful in the present invention.

In some embodiments, the present invention provides a composition whereless than about 5% of the compound is metabolized to rapamycin. In someother embodiments, less than about 1% of the compound is metabolized torapamycin. In still other embodiments, less than about 0.1% of thecompound is metabolized to rapamycin.

In other embodiments, the present invention provides a composition in adosage form, having a daily systemic dose of the compound of from about0.1 mg to about 20 mg. In some other embodiments, the daily systemicdose of the compound is from about 0.5 mg to about 10 mg. In anotherembodiment, the daily systemic dose of the compound is from about 1 mgto about 5 mg.

IV. Treatment

The compounds of the present invention can be used to treat conditionsresponsive to the class of compounds commonly known as macrocyclictrienes or macrocyclic lactones.

The compounds of the present invention can be used to treat diseases inmammals alone or in combination with other agents, including conditionssuch as:

a) Treatment and prevention of acute or chronic organ or tissuetransplant rejection, e.g. for the treatment of recipients of heart,lung, combined heart-lung, liver, kidney, pancreatic, skin or cornealtransplants. They can also be used for the prevention ofgraft-versus-host disease, such as following bone marrowtransplantation.

b) Treatment and prevention of transplant vasculopathies, e.g.atherosclerosis.

c) Treatment and prevention of cell proliferation and migration leadingto vessel intimal thickening, blood vessel obstruction, obstructivevascular atherosclerosis, restenosis.

d) Treatment and prevention of autoimmune disease and of inflammatoryconditions, such as inflammatory conditions with an etiology includingan autoimmune component such as arthritis (for example rheumatoidarthritis, arthritis chronica progrediente and arthritis deformans) andrheumatic diseases.

e) Treatment and prevention of asthma.

f) Treatment of multi-drug resistance conditions such as multidrugresistant cancer or multidrug resistant AIDS.

g) Treatment of proliferative disorders, e.g. tumors, cancer,hyperproliferative skin disorder and the like.

h) Treatment of infections such as fungal, bacterial and viral.

i) Treatment or prevention of cellular proliferation in vascular shunts.

j) Treatment or prevention of ophthalmic conditions and diseases.

k) Prevention of neo-vascularization.

The potency of Compound AR and rapamycin (Sirolimus) for inhibitinghuman cell proliferation is demonstrated in an in vitro model. The testis described in Example 2 and the results are shown in FIG. 8. CompoundAR inhibited growth of smooth muscle cells over a range ofconcentrations as demonstrated in FIG. 8.

In some embodiments, the present invention provides a method ofinhibiting cell proliferation or migration by administering to a subjectin need thereof, a therapeutically effective amount of a compound of thepresent invention.

In other embodiments, the present invention provides a method whereinthe compound of the present invention is administered systemically,locally or via a combination thereof.

In some other embodiments, the administration of the compound of thepresent invention is via oral administration, administration as asuppository, topical contact, parenteral, intravascular, intravenous,intraperitoneal, intramuscular, intralesional, intranasal, pulmonary,mucosal, transdermal, ophthalmic, subcutaneous administration orintrathecal administration.

In still other embodiments, the administration of the compound of thepresent invention is via delivery through a temporary device or animplant. In another embodiment, the temporary device is selected fromthe group consisting of a catheter and a porous balloon. In stillanother embodiment, the implant is a vascular prosthesis. In yet otherembodiments, the vascular prosthesis comprises an expandable structure.In another embodiment, the vascular prosthesis comprises a stent, graft,or a scaffold formed at least in part from an open lattice.

In one embodiment, the inhibition concentration (IC₅₀) of compounds ofthe present invention is approximately equal to the IC₅₀ of itscorresponding parent macrocyclic lactone (prior to the modifications inFIG. 1). In another embodiment, the IC₅₀ is higher than the IC₅₀ of itscorresponding parent macrocyclic lactone. In yet another embodiment, theIC₅₀ is lower than the IC₅₀ of its corresponding parent macrocycliclactone. For example, the IC₅₀ is two to thousand times lower than theIC₅₀ of its corresponding parent macrocyclic lactone.

In a preferred embodiment, the IC₅₀ of a compound of the presentinvention is 1.5 to 1,000 times higher than its corresponding parentmacrocyclic lactone, preferably 2 to 100 times higher than thecorresponding parent macrocyclic lactone and more preferably 5 to 50times higher than the corresponding parent macrocyclic lactone. Inanother embodiment the IC₅₀ of a compound of the present invention isfrom about 0.1 nM to about 1 μM, preferably from about 1 nM to about 0.5μM, more preferably from about 5 nM to about 100 nM.

Other means of measuring the effectiveness of the compounds of thepresent invention include measuring effective concentration (EC₅₀). Inone embodiment, the EC₅₀ is approximately equal to the EC₅₀ of thecorresponding parent macrocyclic lactone. In another embodiment, theEC₅₀ is higher than the EC₅₀ of the corresponding parent macrocycliclactone. In yet another embodiment, the EC₅₀ is lower than the EC₅₀ ofthe corresponding parent macrocyclic lactone.

In some embodiments, the present invention provides a method wherein theeffective dose of the compound is from about 0.1 mg to about 20 mg. Insome other embodiments, the effective dose of the compound is from about0.5 mg to about 10 mg. In still other embodiments, the effective dose ofthe compound is from about 1 mg to about 5 mg.

The compounds, compositions and devices of the present invention areuseful for cytokine inhibition. Pro-inflammatory cytokine IL-6 issynthesized in response to diverse inflammatory stimuli and acts as akey regulatory protein in the inflammatory cascade. IL-6 plays a pivotalrole in stimulating the acute-phase response after injury, including therelease of fibrinogen and C-reactive protein.

IL-6 may also be directly involved in restenosis, as it has been shownto stimulate leukocyte recruitment into the vessel wall, and vascularsmooth muscle cell proliferation, factors that are essential to thepathogenesis of hyperproliferative diseases such as restenosis.

Matrix metalloproteinases (MMP-9) play a key role in cellular migrationand proliferation including conditions such as neointimal growth andvascular remodeling after stent implantation. Release of MMPs causeincreases in proteoglycan rich, extracellular matrix which increasessmooth muscle cell migration after vascular injury.

Plasma active MMP-9 levels may be a useful independent predictor of baremetal stent ISR. (Elevated Plasma Active Matrix Metalloproteinase-9Level Is Associated With Coronary Artery In-Stent Restenosis,Arterioscler Thromb Vasc Biol. 2006;26:e121-e125.)

Monocyte chemoattractant protein 1 (MCP-1) is a potent monocytechemoattractant secreted by many cells in vitro, including vascularsmooth muscle and endothelial cells. Eliminating MCP-1 gene or blockadeof MCP-1 signals has been shown to decrease atherogenesis inhypercholesterolemic mice. MCP-1 has been shown to play a role inpathogenesis of neointimal hyperplasia in monkeys. (Importance ofMonocyte Chemoattractant Protein-1 Pathway in Neointimal HyperplasiaAfter Periarterial Injury in Mice and Monkeys, Circ Res.2002;90:1167-1172.) MCP-1 is also strongly expressed in a small subsetof cells in macrophage-rich regions of human and rabbit atheroscleroticlesions (Expression of Monocyte Chemoattractant Protein 1 inMacrophage-Rich Areas of Human and Rabbit Atherosclerotic Lesions, PNAS,Vol 88, 5252-5256) Inhibition of MCP-1 can have therapeutic impact ontreatment and prevention of inflammatory, proliferative and otherdisease conditions discussed above.

Interleukin-10 (IL-10) is an anti-inflammatory cytokine with a powerfulinhibitory effect on monocytes. IL-10 has been shown to reducepostinjury intimal hyperplasia (Interleukin-10 Inhibits IntimalHyperplasia After Angioplasty or Stent Implantation inHypercholesterolemic RabbitsCirculation. 2000;101:908-916). Endogenousproduction of IL-10 by human monocytes in response to LDL stimulationinhibits IL-12 production, indicating a cross-regulatory action of IL-10that may counterbalance the proinflammatory response.

A. Ophthalmic Conditions and Diseases

In some embodiments, the compounds, pharmaceutical compositions anddevices of the present invention are useful for the treatment ofophthalmic conditions and diseases. The compounds, pharmaceuticalcompositions and devices of the present invention are useful in thetreatment of any ophthalmic condition or disease. Ophthalmic conditionsand diseases that can be treated by the compounds and devices of thepresent invention include, but are not limited to, disorders of theeyelid, disorders of the lacrimal system and orbit, tear duct blockage,disorders of conjunctiva, disorders of the sclera, cornea, iris andciliary body, disorders of the lens, disorders of the choroid andretina, Age-related Macular Degeneration (AMD), Diabetic Macular Edema(DME), glaucoma, disorders of the vitreous body and globe, disorders ofthe optic nerve and visual pathways, disorders of the ocular muscles,binocular movement, accommodation and refraction, visual disturbancesand blindness, etc. Additional ophthalmic conditions and diseases thatcan be treated with the compounds and devices of the present inventioninclude inhibition of cell proliferation, prevention of inflammation,prevention of neovascularization, protection of neurovascular system,and prevention of immune response after transplantation. One of skill inthe art will appreciate that other ophthalmic conditions and diseasescan be treated using the compounds and devices of the present invention.

Current treatment methods include surgery and medications. Surgicaltreatment methods include retinal implant, high speed laser eye surgery,endothelial keratoplasty, cataract surgery, glaucoma surgery, refractivesurgery, corneal surgery, vitreo-retinal surgery, eye muscle surgery,oculoplastic surgery, uses of stem cells to create corneas or part ofcorneas that can be transplanted into the eyes.

Ophthalmic conditions and diseases can be treated using compounds,pharmaceutical compositions and devices of the present invention, asdescribed above. The compounds and pharmaceutical compositions of thepresent invention can be administered via any method known to one ofskill in the art. In some embodiments, the compounds of the presentinvention are administered via implant, injection or eye drop. In someother embodiments, the administration is through an intraocular orintravitreal body of the eye. In other embodiments, the administrationis via the implant. In other embodiments, the compounds are administeredvia an implant where the compound is released via a metallic, ceramic orpolymer coating.

When administration is via the implant, the compound can be released byany means known in the art. In some embodiments, release of the compoundfrom the implant can be via osmotic pressure of diffusion. One of skillin the art will appreciate that other means of releasing the compoundfrom the implant are useful in the present invention.

In some embodiments, the compounds of the present invention are combinedwith at least one other therapeutic agent for treatment of an ophthalmiccondition or disorder. Any suitable therapeutic agent known to one ofskill in the art can be combined with the compounds of the presentinvention for use in the treatment of ophthalmic conditions or diseases.In some embodiments, the therapeutic agents include, but are not limitedto, anti-inflammatory agents, immunomodulating agent and anti-infectiveagents, antioxidants, antibody, antibiotics, anti-angiogenics,anti-vascular endotherlial growth factor agent, antihistamines andlubricant. Therapeutic agents that can be combined with the compounds ofthe present invention include, but are not limited to, lucentis,avastin, macugan, volociximab, olopatadine, mydriatcs, dexamethasone,pilocarpine, tropicamide, quinolone, galentamine, fluocinoloneacetonide, triamcinolone acetonide, atropine, atropine sulfate, atropinehydrochloride, atropine methylbromide, atropine metliylnitrate, atropinehyperduric, atropine N-oxide, phenylephrine, phenylephrinehydrochloride, hydroxyamphetamine, hydroxyamphetamine hydrobromide,hydroxyamphetamine hydrochloride, hydroxyamphetamine iodide,cyclopentolate, cyclopentolate hydrochloride, homatropine, homatropinehydrobromide, homatropine hydrochloride, homatropine methylbromide,scopolamine, scopolamine hydrobromide, scopolamine hydrochloride,scopolamine methylbromide, scopolamine methylnitrate, scopolamineN-oxide, tropicamide, tropicamide hydrobromide, tropicamidehydrochloride, pilocarpine, isopilocarpine, physostigmine, andquaternary ammonium compounds including stearyl ammonium chloride andbenzyl ammonium chloride, including mixtures, ionic salts, andcombinations thereof.

It can be appreciated that all embodiments disclosed in the presentinvention can be utilized alone or in combination with other embodimentsor examples in this invention.

V. Example

Example 1: Preparation of 16-O-demethyl Macrocyclic Lactone (CompoundAR)

Macrocyclic lactone rapamycin (1000 mg, 10.75 mmol) in 500 mlacetonitrile was treated with 500 ml 0.1N hydrochloric acid. Theresulting solution was stirred at room temperature for about 28 hours.Then the reaction mixture was extracted with dichloromethane in aseparatory funnel. The organic layers were washed with water and brine,then washed with 0.1M sodium phosphate buffer (pH=7.4) twice or untilpH=7, then washed three times with distilled (DI) water. Finally theorganic layer was dried over Na₂SO₄ and placed in a refrigeratorovernight. Concentration in vacuum provided an off-white powder ofCompound AR (approx. 820 mg).

Compound AR was purified by preparative HPLC on an Ascentis C18(21.2×250 mm, 10 μm) column from SUPECO using Methanol:water (80:20) asmobile phase from 0-12 min then changed to 100% Methanol from12.01-20min at a flow rate of 15 ml/min. The loading concentration was350 mg/ml and the injection volume was 100 ul. The compound wasmonitored by UV absorbance at 254 nm. Under these conditions, CompoundAR eluted between 9.0-11.5 min whereas the starting materials and byproducts eluted between 17.0-20.0 min.

The preparative HPLC can also be conducted using a gradient ofacetonitrile:water (starting from 70:30) as mobile phase and monitoredby UV absorbance at 278 nm. The preparative HPLC chromatogram using thismethod of separation is shown in FIG. 4.

The fractions containing Compound AR were collected and pooled together,then solvent was evaporated by using a rotovac and freeze drier to givean off-white powder of Compound AR.

¹H NMR (CDCl₃, 400 MHz, mixture of trans:cis amide rotamers, chemicalshifts in parentheses refer to the major rotamer) is presented in FIG.5. δ, ppm 0.532 (q, J=12 Hz, 1H), 0.890 (d, J=6.8 Hz, 3H), 0.921 (d,J=6.8 Hz, 3H), 0.931 (d, J=6.4 Hz, 3H), 0.971 (d, J=6.8 Hz, 3H), 0.991(d, J=6.6 Hz, 3H), 1.005 (d, J=6.4 Hz, 3H), 1.686 (s, 3H), 1.772 (s,3H), 1.773 (s, 3H), 1.823 (s, 3H), 3.330 (s, 3H), 3.380 (s, 3H), 3.859(d, J=5.2 Hz, 1H), 4.001 (d, J=3.6 Hz, 1H), 4.03-4.07 (m, 1H), 4.22 (br,1H), 5.21-5.28 (m, 3H), 5.336 (d, J=11.6 Hz, 1H), 5.384 (dd, J=14.8, 9.6Hz, 1H), 6.117 (dd ,J=14.4, 10.8 Hz, 1H), 6.243 (dd ,J=14.4, 10.4 Hz,1H), 6.376 (dd, J=14.8,11.2 Hz, 1H).

Compared with the proton NMR of the parent compound, the disappearanceof the peak at 3.14 ppm demonstrates the demethylation at the C16position only and completion of reaction. (See Journal of Antibiotics1991, 44(6), 688 for assignment of the NMR spectra for rapamycin.)

The chemical structure of Compound AR was further verified by massspectrometric experiments. The fragmentation patterns indicated thepresence of the m/z 900 whereas Rapamycin provides m/z 914 under thesame conditions. The results of liquid chromatography and massspectroscopy experiments are provided in FIG. 6 which shows theidentification of Compound AR with m/z 900.

The total content of Compound AR was determined by a reverse phase HPLCusing a Supelco C18 (4.6×150 mm, 5 μm) column from Sigma Aldrich usingmethanol:water (90:10) as mobile phase and a flow rate of 1 ml/min.Compound AR was monitored by UV absorbance at 254 nm. Compound AR had aretention time of 7.97 min. FIG. 7 shows the analytical HPLCchromatogram of Compound AR with total content of >98%. The purity ofCompound AR was determined by a reverse phase HPLC using a YMC ODS-ALC18 (4.6×250 mm, 5 μm) column from Waters Corporation using anacetonitrile:water gradient mobile phase and a flow rate of 1.0 ml/min.FIG. 7 b shows major isomers of Compound AR with a purity >98% asmonitored by UV absorbance at 278 nm.

As a means to minimize oxidation of the Compound AR, 0.1% w/w ofButylated Hydroxytoluene (BHT) was added after preparative HPLC.

Example 2: Biological Activity of Compound AR

Potency of the Compound AR was demonstrated by in vitro human smoothmuscle cell culture testing. The amounts of incorporated thymidine forsamples of Compound AR of varying concentrations (0.005, 0.01, 0.05,0.1, 0.5, and 1 μM) and of rapamycin of varying concentrations (0.0005,0.001, 0.01, and 0.1 μM) were measured after exposure for differenttimes of 1, 3 and 8 hours. The IC₅₀ for Compound AR and rapamycin wereapproximately 0.05 and 0.01 μM, respectively, after smooth muscle cellswere exposed to Compound AR and rapamycin for the shorter periods of 1and 3 hours (as shown in Table 1 and FIG. 8). The IC₅₀ of Compound ARand rapamycin was approximately 0.005 and 0.001 μM, respectively, aftersmooth muscle cells were exposure to Compound AR and rapamycin for 8hours. The IC₅₀ of Compound AR was approximately five times higher thanthat of rapamycin.

TABLE I Data of percentage proliferation of human smooth muscle cellsafter exposure to varying concentrations of rapamycin and Compound ARCompound AR Concentration (micromolar) 0.005 0.01 0.05 0.1 0.5 %Proliferation After One Hr Exposure to 84 66 54 55 57 Compound AR %Proliferation After Three Hr Exposure 69 56 50 52 48 to Compound AR %Proliferation After Eight Hr Exposure 52 38 40 36 35 to Compound ARRapamycin Concentration (micromolar) 0.0005 0.001 0.01 0.1 %Proliferation After One Hr Exposure to 105 75 54 50 Rapamycin %Proliferation After Three Hr Exposure to 79 63 48 39 Rapamycin %Proliferation After Eight Hr Exposure to 57 51 40 38 Rapamycin

Example 3: Preparation of Stents Containing Compound AR

15 mg poly(n-butyl methacrylate) (PBMA) was dissolved into 3 mLdichloromethane at room temperature. 10 mg of Compound AR was placed ina vial and dissolved in 2 mL dichloromethane with or without 0.1% (w/w)BHT. The solutions were combined and further diluted with 10 mLdichloromethane.

A microprocessor-controlled ultrasonic sprayer was used to apply 450 ugof the drug containing PBMA solution to the entire surface of a 18 mmmetal stent (available from Elixir Medical Corp, Sunnyvale, Calif.).After coating, the stent was placed in a vacuum chamber. The stent wasthen mounted on the balloon of a 3.0×20 mm PTCA delivery catheter. Thecatheter was then inserted in a coil and packaged in a Tyvek® pouch. Thepouch was sterilized by ethylene oxide. The Tyvek® pouch was furtherpackaged in a foil pouch with oxygen scavengers and nitrogen purge andvacuum sealed.

Example 4: In vivo Testing of Stents Eluting Compound AR

The efficacy of a Compound AR eluting stent system (as prepared above)from Example 3 was evaluated by comparing 28±2 day angiographic outcomesin porcine coronary arteries to the rapamycin eluting stent system,Cypher™ Coronary Stent (Cordis Corporation) in the non-diseased porcinecoronary artery model.

The nonatherosclerotic swine model was chosen as this model has beenused extensively for stent and angioplasty studies resulting in a largevolume of data on the vascular response properties and its correlationto human vascular response (Schwartz et al, Circulation.2002;106:1867-1873). The animals were housed and cared for in accordancethe Guide for the Care and Use of Laboratory Animals as established bythe National Research Council.

All animals were pretreated with aspirin (325 mg) and clopidogel (75 mg)per oral dose beginning at least 3 days prior to the intervention andcontinuing for the duration of the study. After induction of anesthesia,the left or right femoral artery was accessed using standard techniquesand an arterial sheath was introduced and advanced into the artery.

Vessel angiography was performed under fluoroscopic guidance, a 7 Fr.guide catheter was inserted through the sheath and advanced to theappropriate location where intracoronary nitroglycerin was administered.A segment of coronary artery ranging from 2.25 to 4.0 mm mean lumendiameter was chosen and a 0.014″ guidewire inserted. QuantitativeCoronary Angiography (QCA) was performed to document the referencevessel diameter.

The appropriately sized stent was advanced to the deployment site. Theballoon was inflated at a steady rate to a pressure sufficient toachieve a balloon to artery ratio of 1.30:1.0. Pressure was maintainedfor approximately 10 seconds. Angiography was performed to documentpost-procedural vessel patency and diameter.

Follow-up angiography was performed at the designated endpoint for eachof the animals. Each angiogram was qualitatively evaluated for evidenceof stent migration, lumen narrowing, stent apposition, presence ofdissection or aneurysms, and flow characteristics. Upon completion offollow-up angiography, the animals were euthanized.

The hearts were harvested from each animal and the coronary arterieswere perfused with 10% buffered formalin at 100 to 120 mm Hg. The heartswere immersed in 10% buffered formalin. Any myocardial lesions orunusual observations were reported.

Angiographic parameters measured or calculated included:

Marginal vessel (proximal and distal) mean lumen diameter (post-stentand final only)

Mean lumen diameter of the target region (all angiograms)

Minimal lumen diameter (MLD) of the target region (post-stent and finalonly)

Diameter stenosis [1−(MLD/RVD)]×100% where RVD is a calculation of thereference diameter at the position of the obstruction (measure obtainedby a software-based iterative linear regression technique to generate anintrapolation of a projected vessel without the lesion) (final angiogramonly)

Balloon to artery ratio [balloon/pre-stent mean luminal diameter]

Stent to artery ratio [post-stent/pre-stent mean luminal diameter]

Late loss ratio [MLD final-MLD post-stent]

All animal survived to the designated end point. There were nodocumented incidents of stent migration, stent malapposition, persistentdissection or evidence of aneurysm. Three outlying data points (totalocclusion or near total occlusion) for the Cypher

Stent were excluded. The average percent stenosis for the Compound ARstent (approx. 10 microgram/mm length drug dose) was 25.7±17.8 (n=15) ascompared to Cypher Stent pooled data from this and previous studies withsimilar protocols which provided an average percent stenosis of20.21±11.45 (n=22) for Cypher stents. (FIG. 9).

The Compound AR eluting stents in this example when implanted in theporcine model for 28 days resulted in higher percentage stenosis ascompared to the Cypher Stent.

Example 5: In vivo Pharmacokinetics of Stents Eluting Compound AR

Pharmacokinetic evaluation of the Compound AR stent system from Example3 was performed at 6 hours, 3 days, 7 days, and 28 days in the porcinecoronary artery model. The interventional procedures used were similarto the in vivo angiographic study described in Example 4 up to stentimplantation.

The appropriately sized stent was advanced to the deployment site. Theballoon was inflated at a steady rate to a pressure sufficient toachieve a balloon to artery ratio of 1:1. Pressure was maintained forapproximately 10 seconds. Angiography was performed to documentpost-procedural vessel patency and diameter. A total of 9 stents (3 pertime point) were implanted.

At the appropriate time point the animals were euthanized and the heartsexcised. The stented segment including approximately 10 mm of vesselproximal and 10 mm distal to the stented section was excised. Theproximal and distal sections were separated and stored in separatevials. The tissue surrounding the stent was carefully removed from stentand each place in separate vials. All were then frozen to −70° C. priorto being analyzed using liquid chromatography mass spectroscopy (LCMS).

All animal survived to the designated end point. The average tissueconcentrations for the Compound AR and stent release rates are presentedin FIGS. 10 and 11. The Compound AR eluting stent, in this exampledemonstrates release of Compound AR from the stent with greater than 40%of the drug released at 7 days.

Example 6: Preparation of 17, 18-29, 30-bis-epoxide Macrocyclic Lactone(AS)

Add 0.8 mL 5% NaOH—MeOH and 2 ml 30% H₂O₂ to a solution of 1 gramRapamycin in 40 ml methanol. The reaction mixture was stirred at roomtemperature for 24 hours. If TLC indicated some rapamycin was stillunreacted, additional mixture of 0.8 mL 5% NaOH—MeOH and 2 ml 30% H₂O₂was added to the reaction solution. Stirring continued at roomtemperature until TLC indicated the reaction has completed. The solutionwas extracted with dichloromethane and brine 3 times. The organic layerswere combined and washed with brine and water, dried over anhydrousMgSO4, filtered MgSO4 and the solution was evaporated to leave a rawproduct. This raw product was further purified using TLC plate andgiving 0.40 g (40% yields) light yellow powder. ¹H NMR (CDCl₃) (˜4:1mixture of conformers, only signals of major conformer listed) Majorchanges compared with rapamycin δ (ppm) 1.75(s, 1H), 1.98(s, 1H),6.71(ddd, 5H, J1=16 Hz, J2=8 Hz, J3=2.8 Hz). ¹³C DEPT 135 NMR (CDCl₃)(4:1 mixture of conformers, only signals of major conformer listed) δ(ppm) 152, 140, 133, 128, 127, 126, 84, 83, 76, 74, 67, 59, 56, 44, 43,41, 40, 36, 35, 34, 33, 31, 29, 28, 22, 21, 20, 17, 16, 15, 13. MassSpectra m/z=962 with Rapamycin m/z=930.

Example 7: Biological Activity of 17, 18-29, 30-bis-epoxide MacrocyclicLactone (AS)

Potency of the 17, 18-29, 30-bis-epoxide macrocyclic lactone wasdemonstrated by in vitro human smooth muscle cell culture testing. Theamounts of incorporated thymidine for samples of 17, 18-29,30-bis-epoxide macrocyclic lactone of varying concentrations (0.005,0.01, 0.05, 0.1, 0.5, and 1 μM) and of Compound AR of varyingconcentrations (0.0005, 0.001, 0.01, and 0.1 μM) were measured afterexposure for 8 hours. The IC₅₀ of 17, 18-29, 30-bis-epoxide macrocycliclactone and Compound AR was approximately 0.1 and 0.005 μM,respectively, after smooth muscle cells were exposed to 17, 18-29,30-bis-epoxide macrocyclic lactone and Compound AR for 8 hours (FIG.13). The IC₅₀ of 17, 18-29, 30-bis-epoxide macrocyclic lactone wasapproximately 20 times higher than that of Compound AR.

Example 8: Preparation of 17, 18-19, 20-21, 22-tris-epoxide MacrocyclicLactone (AY)

m-chloro-peroxybenzoic acid 0.93 g (3.22 mmol) was added to a solutionof 0.50 g (0.5371 mmol) rapamycin in 10 ml CHCl₃ at room temperature.The mixture was stirred at room temperature for 24 hours. If TLCindicated some rapamycin was still unreacted, additionalm-chloroperoxybenzoic acid 0.50 g and 5 mL CHCl₃ was added. Stirring wascontinued at room temperature until no Rapamycin was indicated by TLC.After completion of the reaction, the solution was diluted withdichloromethane, treated with aqueous sodium sulfite until the washingsgive a negative test with starch-iodide paper. This ensures all excessperacid has been destroyed and the aqueous layer was extracted withseveral portions of CH₂Cl₂. Then organic layer was washed with two 20 mlportion of 5% sodium bicarbonate to remove benzoic acid. The combinedorganic extracts were washed with water, dried with anhydrous MgSO₄,filtered and evaporated to leave a crude white solid 0.46 g. The rawproduct was further purified using TLC plate. MS m/z 978 with Rapamycinm/z 930.

Example 9: Cytokine Inhibition by Macrocyclic Lactone:

In cell culture studies, macrophages were activated to secrete cytokinessuch as Il-6, MMP-9, MCP-1 and IL-10 by treating the cells to E Colilippopolysaccharide (LPS). Inhibition of these cytokines upon treatmentof the activated macrophages with Compound AR and rapamycin with 10 nMconcentration was tested using ELISA assay. The inhibition ofpro-inflammatory and cell proliferation and migration inducing cytokinesupon exposure to the macrocyclic lactone is presented in FIG. 12( a).The inhibition of anti-inflammatory cytokine IL-10 upon exposure tomacrocyclic lactone Compound AR and Sirolimus are presented in FIG. 12(b).

MMP-9 levels in the 1st, 3rd and 7th day after stent implantation werepositively correlated to the late loss index 6 months after stentimplantation (Elevated matrix metalloproteinase expression after stentimplantation is associated with restenosis. Int J Cardiol. 2006;112(1):85-90).

Compound AR and Sirolimus did not show and significant inhibition ofrelease of IL-6. Compound AR significantly reduced the production ofboth the cytokines MMP-9 and MCP-1 as compared to Sirolimus whichincreased the production of MMP-9 and did not impact the production ofMCP-1.

Compound AR and Sirolimus did not show any difference in inhibition ofrelease of IL-6. On the other hand, Compound AR reduced the productionof cytokine MMP-9 while Sirolimus increased the production of MMP-9.Compound AR reduced the production of cytokine MCP-1 as compared toSirolimus which did not impact the production of MCP-1. Compound AR andSirolimus both inhibit the production of anti-inflammatory cytokineIL-10.

Compounds claimed in the present invention, such as Compound AR canprovide better therapeutic response with higher levels ofanti-inflammatory effect (such as greater inhibition of pro-inflammatorycytokine MCP-1) and higher levels of anti-cell proliferative andanti-cell migratory effect (such as greater inhibition ofpro-proliferative and migration cytokine MMP-9).

Example 10: Testing of Compound AR Eluting Stents in Human ClinicalTrial:

Clinical testing of the Compound AR coated stent was conducted on 15human subjects. Safety of the Compound AR coated stent was evaluatedclinically through the evaluation of major adverse cardiac eventsdefined as: death, myocardial infarction (both Q-wave and non-Q-wave),and target lesion revascularization. Efficacy was evaluated throughangiographic and intravascular ultrasound (IVUS) results at 4 months.The primary endpoint of the study was angiographic in-stent late lumenloss. Secondary endpoints were Major Adverse Cardiac Events (MACE) andadditional angiographic and IVUS evaluation. The clinical study wasapproved by local Ethics Committee and all patients signed an Ethicsapproved informed consent before entry into the clinical study.

All patients were pretreated with aspirin and ticlopidine (500 mg) peroral beginning at least 1 day prior or on the day of the indexprocedure. Aspirin (>100 mg/day and Clopidogrel (75 mg/day) werecontinued through for at least six months. In accordance with hospitalstandard percutaneous practice, the left or right femoral artery wasaccessed using standard techniques and an arterial sheath was introducedand advanced into the artery.

Index procedure vessel angiography was performed under fluoroscopicguidance, a 6 or 7 Fr. guide catheter was inserted through the sheathand advanced to the appropriate location; intracoronary nitroglycerinwas administered. A segment of coronary artery ranging from 3.0 mm to3.5 mm mean lumen diameter was chosen and a 0.014″ guidewire inserted.Quantitative Coronary Angiography (QCA) was performed to document thereference vessel diameter. Predilatation of the lesion was performedprior to stent implantation using standard technique.

Following predilatation, the appropriately sized stent (3.0×18 mm or3.5×18 mm was advanced to the deployment site. The balloon was inflatedat a steady rate to a pressure to fully deploy the stent. Pressure wasmaintained for approximately 30 seconds. Post dilatation of the stentcould be performed as needed to assure good stent apposition to thevessel wall. Angiographic and intravascular ultrasound imaging (IVUS)was performed and recorded.

Follow-up angiography and IVUS was performed at the designated endpointof 4 months for each patient. Each angiogram was qualitatively evaluatedfor evidence of lumen narrowing, stent apposition, and flowcharacteristics.

Angiographic and IVUS parameters measured or calculated included:

Marginal vessel (proximal and distal) mean lumen diameter (post-stentand final)

Mean lumen diameter of the target region (all angiograms)

Minimal lumen diameter (MLD) of the target region (post-stent and finalonly)

Diameter stenosis [1−(MLD/RVD)]×100% ] where RVD is a calculation of thereference diameter at the position of the obstruction (measure obtainedby a software-based iterative linear regression technique to generate anintrapolation of a projected vessel without the lesion) (final angiogramonly).

In-stent Late Lumen Loss [MLD final-MLD post-stent]

In-stent percent neointimal volume as assessed by IVUS

All patients underwent 4 month clinical and angiographic follow-up. Nopatients experience any major adverse cardiac events during thefollow-up period. Angiographic results demonstrated that the primaryendpoint of angiographic in-stent late lumen loss was 0.16±0.32 mm. IVUSanalysis was conducted on 13 of 15 patients and the results demonstratedin-stent percent neointimal volume of 3.7±2.7%.

As a comparison, Cypher stent tested in a pilot study and demonstratedsimilar clinical safety with no clinical events and angiographic resultsat 4 months of in-stent late lumen loss for the slow release group (thecurrent commercially available formulation) of 0.09±0.3 mm and in-stentpercent neiontimal volume by IVUS to be 0.3±0.6% (Sousa, J E,Circulation 2001;103;192-195).

Example 11: Preparation of 31-hydroxyl, 44-hydroxyl (Compound BG) and47-hydroxyl (Compound BJ) Macrocyclic Lactone

Macrocyclic lactone rapamycin (1 g, 1.1 mmol) in 100 ml absolute ethanolwas added to selenium dioxide (122 mg, 1.10 mmol) absolute ethanol (50ml) solution. The mixture was stirred at room temperature until TLCanalysis showed the disappearance of rapamycin (about 15 h). Thereaction mixture was concentrated and the residue was partitionedbetween ether and water and extracted with ether 3×80 ml. The organiclayers was dried over anhydrous sodium sulfate, filtered andconcentrated. The final individual pure product 31-hydroxyl,44-hydroxyl, 47-hydroxyl macrocyclic lactone were obtained by preparedHPLC.

The characterized spectra changes compared with macrocyclic lactonerapamycin are shown as follows:

Sample name 44-hydroxyl 47-hydroxyl 31-hydroxyl macrocyclic macrocyclicmacrocyclic lactone lactone lactone (compound BG) (compound BJ) Mass M/Z929 929 929 [M + H] ¹HNMR (1:1 Loss of 0.87, Loss of 1.58 ppm Loss of1.72 ppm THF-d₈/D₂O) 3.21, 3.27 ppm single peak; single peak; peak; Gain1.38 Gain 4.07 Gain 4.07 ppm peak ppm peak ppm peak

Example 12: Preparation of 43-hydroxyl(Compound BF),47-hydroxyl(Compound BJ) Macrocyclic Lactone

A solution of 1 gram macrocyclic lactone rapamycin (1.1 mmol) wasdissolved in 100 ml acetonitrile. 0.3 gram Ferrous Sulfate heptahydrate(1.1 mmol) was added to the solution with stirring and nitrogen purgingfor 20-30 minutes. The mixture was then cooled to −16° C. to −18° C.with ice-bath. A solution of 1 ml 30% H₂O₂ in 10 ml acetonitrile wasthen slowly added to above mixture over 30 min with stirring andnitrogen purging. After complete addition of hydrogen peroxide,continued stirring and nitrogen purging until TLC analysis showed thedisappearance of rapamycin. The solution was extracted withdichloromethane and brine 3 times. The organic layer was combined andwashed with brine and water, dried over anhydrous MgSO₄, filtered MgSO₄and the solution was evaporated to leave a raw product. The pure product43 -hydroxyl, 47-hydroxyl macrocyclic lactone were obtained by preparedHPLC.

The characterized spectra changes compared with macrocyclic lactonerapamycin are shown as follows:

Sample name 43-hydroxyl 47-hydroxyl macrocyclic macrocyclic lactonelactone (compound BF) (compound BJ) Mass M/Z 929 929 [M + H] ¹HNMR, (1:1THF- Loss of 0.74 ppm peak; Loss of 1.72 ppm single d₈/D₂O) Gain 3.49,3.24 peak; Gain 4.07 ppm peaks ppm peak

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference.

What is claimed is:
 1. A pharmaceutical composition comprising apharmaceutically acceptable excipient and a compound of the formula:

wherein R¹, R², R³, R⁵, R⁶, R⁸, M¹, M², M³, M⁴, M⁵, M⁶ and M⁷ are eachindependently a member selected from the group consisting of H, C₁₋₆alkyl, OH and C₁₋₆ hydroxyalkyl; R⁴, R⁷ and R⁹ are each independentlyselected from the group consisting of C₁₋₆ alkoxy and OH; R¹⁰ is amember selected from the group consisting of H, —OH, —OP(O)Me₂,

—O—(CH₂)_(n)—OH and —O—(CH₂)_(m)—O—(CH₂)_(o)—CH₃, wherein subscripts nand m are each independently from 2 to 8 and subscript o is from 1 to 6;each of L¹ and L⁴ are independently selected from the group consistingof:

wherein each M⁸ is independently a member selected from the groupconsisting of C₁₋₆ alkyl, OH and C₁₋₆ hydroxyalkyl; each of L² and L³are independently selected from the group consisting of:

and salts, hydrates, isomers, metabolites, N-oxides and prodrugsthereof.
 2. The composition of claim 1, wherein the compound is acompound of FIG.
 2. 3. The composition of claim 1, wherein R¹, R⁶ and R⁸are each methyl; R², R³ and R⁵ are each H; R⁴ and R¹⁰ are each OH; R⁷and R⁹ are each OMe; M¹, M², M³, M⁴ and M⁵ are each H; M⁶ and M⁷ areeach methyl; L¹ and L⁴ are each

and L² and L³ are each


4. The composition of claim 1, wherein the pharmaceutically acceptableexcipient is a member selected from the group consisting of a polymer, asolvent, an antioxidant, a binder, a filler, a disintegrant, alubricant, a coating, a sweetener, a flavor, a stabilizer, a colorant, ametal, a ceramic and a semi-metal.
 5. The composition of claim 4,wherein the pharmaceutically acceptable excipient is a polymer.
 6. Thecomposition of claim 5, wherein the polymer is selected from the groupconsisting of polyurethane, polyethylene imine, ethylene vinyl alcoholcopolymer, silicone, C-flex, nylons, polyamide, polyimide,polytetrafluoroethylene (PTFE), parylene, parylast, poly(methacrylate),poly(vinyl chloride), poly(dimethyl siloxane), poly(ethylene vinylacetate), polycarbonate, polyacrylamide gels, poly (methylmethacrylate), poly(n-butyl methacrylate), poly (butyl methacrylate)copolymer or blended with poly(ethylene vinyl acetate), poly(methylmethacrylate), poly (2-hydroxy ethyl methacrylate), poly(ethylene glycolmethacrylates), poly(ethylene carbonate), Poly L lactide-glycolidecopolymer, poly L lactide-trimethylene carbonate copolymer and PolyL-lactide.
 7. The composition of claim 6, wherein the polymer ispoly(n-butylmethacrylate).
 8. The composition of claim 6, wherein thecompound is present in an amount of at least 25% (w/w) in a mixture ofthe compound and the polymer.
 9. The composition of claim 8, wherein thecompound is present in an amount of at least 50% (w/w).
 10. Thecomposition of claim 8, wherein the compound is present in an amount ofat least 75% (w/w).
 11. The composition of claim 1, wherein less thanabout 5% of the compound is metabolized to rapamycin.
 12. Thecomposition of claim 1, wherein less than about 1% of the compound ismetabolized to rapamycin.
 13. The composition of claim 1, wherein lessthan about 0.1% of the compound is metabolized to rapamycin.
 14. Thecomposition of claim 1 in a dosage form, having a daily systemic dose ofthe compound of from about 0.1 mg to about 20 mg.
 15. The composition ofclaim 14, wherein the daily systemic dose of the compound is from about0.5 mg to about 10 mg.
 16. The composition of claim 14, wherein thedaily systemic dose of the compound is from about 1 mg to about 5 mg.